FACTOR VIII COMPOSITIONSAND METHODS OF MAKING AND USING SAMECROSS-REFERENCE TO RELATED APPLICATION This application claims priority benefit to US. Provisional Application Serial No. ,400filed February 15, 2012, which application is orated herein by reference.
SEQUENCE LISTING The instant ation contains a Sequence Listing which has been submitted in ASCII formatVia EFS-Web, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July11, 2012, is named 32887346.txt and is 13,344,768 Bytes in size.
BACKGROUND OF THE INVENTION Factor VIII is an important component of the intrinsic pathway of the blood coagulationcascade. In the circulation, factor VIII is mainly complexed to von Willebrand factor. Upon activationby thrombin, (Factor Ila), it dissociates from the complex to interact with factor IXa in the intrinsiccoagulation cascade, which, in turn, tes factor X. Once removed from the von Willebrand factorcomplex, activated factor VIII is lytically inactivated by activated Protein C (APC), factor Xa, andfactor IXa, and is quickly cleared from the blood . When complexed with normal von Willebrandfactor protein, the half-life of factor VIII is approximately 12 hours, whereas in the absence of vonWillebrand factor, the ife of factor VIII is d to 2 hours (Tuddenham EG, et al., Br J Haematol.(1982) 52(2):259-267).
In hemophilia, the clotting of blood is disturbed by a lack of certain plasma blood clottingfactors. ilia A is a deficiency of factor VIII, and is a recessive sex-linked, X chromosomedisorder that represents 80% of hemophilia cases. The standard of care for the management ofhemophilia A is replacement therapy with inant factor VIII concentrates. ts with severehemophilia A have circulating procoagulant factor VIII levels below 1-2% of normal and are generallyon prophylactic therapy with the aim of keeping factor VIII above 1% between doses, which can usuallybe achieved by giving factor VIII two to three times a week. Persons with moderately severe hemophilia(factor VIII levels of 2-5% of normal) constitute 25-30% hemophilia incidents and manifest bleedingafter minor trauma. Persons with mild hemophilia A (factor VIII levels of 5-40% of normal) comprise-20% of all hemophilia incidents, and p bleeding only after significant trauma or surgery.
The in vivo activity of exogenously supplied factor VIII is limited both by a short protein half-life and inhibitors that bind to the factor VIII and diminish or destroy atic function.
Up to 30% of hemophilia A patients receiving exogenously-supplied factor VIII mount an IgGimmune se towards factor VIII (Towfighi, F., et al. Comparative measurement of anti-factor VIII 2012/046326antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity. ActaHaematol (2005) 114:84-90), which can result in the te inhibition of its procoagulant activityand/or promote more rapid clearance of the factor VIII (Briet E et al. High titer inhibitors in severehaemophilia A. A meta-analysis based on eight long-term follow-up studies concerning inhibitorsassociated with crude or intermediate purity factor VIII products. Throm. Haemost. (1994) 72: 162-164).
The IgG antibodies, called FVIII inhibitors, are primarily directed towards the A2, A3 and C2 domains(Scandella D et al. zation of epitopes for human factor VIII inhibitor antibodies by immunoblottingand antibody neutralization. Blood (1989) 74:1618-1626), but can arise against the A1, B and C1domains, as well. As such, treatment s for patients with FVIII inhibitors are limited.
Large proteins such as factor VIII are normally given enously so that the medicament isdirectly available in the blood stream. It has been preViously demonstrated that an unmodified factorVIII injected intramuscularly yielded a maximum ating level of only 1.4% of the normal plasmalevel (Pool et al, Ineffectiveness of Intramuscularly Injected Factor VIII Concentrate in Two Hemophilicts. New d I. Medicine (1966) 275(10):547-548). Formulations that could be administeredother than by the intravenous route would y simplify their use, increase safety, and result insubstantial cost savings.
Chemical modifications to a therapeutic protein can modify its in Vivo clearance rate andsubsequent serum half-life. One e of a common modification is the addition of a polyethyleneglycol (PEG) moiety, typically coupled to the n Via an aldehyde or N-hydroxysuccinimide (NHS)group on the PEG reacting with an amine group (e.g. lysine side chain or the inus). However, theconjugation step can result in the formation of heterogeneous product es that require extraction,purification and/or other further processes, all of which ineVitably affect product yield and quality control.
Also, the pharmacologic on of coagulation factors may be hampered if amino acid side chains inthe Vicinity of its binding site become modified by the PEGylation process. Other ches include thegenetic fusion of an EC domain to the therapeutic protein, which increases the size of the therapeuticprotein, hence reducing the rate of clearance through the kidney. In some cases, the Fc domain confersthe ability to bind to, and be recycled from lysosomes by the FcRn receptor, resulting in increasedpharmacokinetic half-life. unately, the Fc domain does not fold efficiently during recombinantexpression, and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodiesmust be solubilized and functional protein must be renatured from the misfolded aggregate, which is atime-consuming, inefficient, and expensive process.
SUMMARY OF THE INVENTION The present invention relates to novel coagulation factor VIII fusion protein compositions andthe uses f. Specifically, the compositions provided herein are ularly used for the treatment orimprovement of a ion associated with hemophilia A, deficiencies of factor VIII, bleeding disordersand coagulopathies. In one aspect, the present invention provides compositions of isolated fusionproteins comprising a factor VIII ) and one or more extended recombinant polypeptides (XTEN) 2012/046326wherein the fusion protein exhibits procoagulant activity. A subject XTEN useful for constructing suchfusion proteins is typically a polypeptide with a non-repetitive ce and unstructured conformation.
In one embodiment, one or more XTEN is linked to a coagulation factor FVIII (“CF”) selected fromnative human factor VIII, factor VIII in d sequences (“FVIII BDD”), and sequence variantsthereof (all the foregoing collectively “FVIII” or “CF”), resulting in a recombinant factor TENfusion protein (“CFXTEN”). The factor VIII polypeptide component of the CFXTEN comprises an Aldomain, an A2 domain, a Cl domain, a C2 domain, and optionally a B domain or a portion f Insome embodiments, the FVIII is further characterized by delineation of the aforementioned domains tose an acidic a1, a2 and a3 spacer. In another embodiment, the present disclosure is directed topharmaceutical compositions comprising the fusion proteins and the uses thereof in methods andregimens for treating factor VIII-related conditions. The CFXTEN compositions have enhancedpharmacokinetic and pharmacologic properties ed to FVIII not linked to XTEN, which maypermit more convenient dosing and improved efflcacy.
In a first aspect, the invention relates to recombinant factor VIII fusion proteins comprising afactor VIII polypeptide and one or more extended recombinant polypeptide (XTEN) linked to the factorVIII. In some embodiments, the invention provides recombinant factor VIII fusion proteins comprising afactor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factorVIII polypeptide comprises an Al domain including an al acidic spacer region, an A2 domain ingan a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, Cl domain, C2 domainand optionally all or a portion of B domain, and wherein said at least one XTEN is linked to said factorVIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) within B domain of said factorVIII polypeptide if all or a portion of B domain is present; (iii) within the Al domain of said factor VIIIpolypeptide; (iV) within the A2 domain of said factor VIII polypeptide; (V) within the A3 domain of saidfactor VIII polypeptide; (Vi) within the Cl domain of said factor VIII polypeptide; (Vii) within the C2domain of said factor VIII polypeptide; (Viii) at the N—terminus of said factor VIII polypeptide, or (ix)between two domains of said factor VIII polypeptide, wherein the fusion protein retains at least about%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% of theprocoagulant ty, when measured by an in Vitro coagulation assay, compared to a correspondingfactor VIII not linked to XTEN. In one embodiment, in the ing recombinant factor VIII fusionn the at least one XTEN is linked to said factor VIII polypeptide at a site at or within 1 to 6 aminoacids of a site selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In other embodiments, theinvention provides recombinant factor VIII fusion ns comprising a factor VIII polypeptide and atleast a first extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprisesan Al domain including an al acidic spacer , an A2 domain including an a2 acidic spacer region,an A3 domain including an a3 acidic spacer region, a Cl domain, a C2 domain and optionally all or an of a B domain, and wherein said first XTEN is linked to said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) within the B domain of said factor VIII ptide if all ora portion of the B domain is present; (iii) within the Al domain of said factor VIII polypeptide; (iV)within the A2 domain of said factor VIII polypeptide; (V) within the A3 domain of said factor VIIIptide; (Vi) within the C1 domain of said factor VIII polypeptide; or (Vii) within the C2 domain ofsaid factor VIII polypeptide; and when ed to a corresponding factor VIII protein not linked toXTEN, the fusion protein (a) retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%100%, 200%, 300%, 400%, or 500% of the procoagulant actiVity in an in Vitro coagulation assaydescribed herein or other such assays known in the art, and/or (b) exhibits reduced binding to an anti-factor VIII antibody in an in vitro binding assay described herein or other such assays known in the art. In one embodiment, in the ing recombinant factor VIII fusion protein the at least one XTEN islinked to said factor VIII polypeptide at a site at or within 1 to 6 amino acids of a site ed from Table, Table 6, Table 7, Table 8, and Table 9. In other ments, the invention provides recombinantfactor VIII fusion proteins comprising a factor VIII polypeptide and at least a first extended recombinantpolypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain including an alacidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3acidic spacer region, a C1 domain, a C2 domain and optionally all or a portion of a B domain, andwherein said first XTEN is linked to said factor VIII polypeptide at an insertion site selected from Table6 and Table 7 and n the fusion protein retains at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%,100%, 200%, 300%, 400%, or 500% of the procoagulant activity, when ed by anin vitro coagulation assay described herein or other such assays known in the art, compared to acorresponding factor VIII protein not linked to XTEN. Non-limiting examples of the factor VIII proteinnot linked to XTEN includes native FVIII, BDD FVIII, pBC100 and ces from Table 1. In anotherembodiment of the recombinant factor VIII fusion protein, the factor VIII polypeptide has at least about80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% ce identity to asequence selected from the group consisting of the sequences of Table 1, the ce depicted in and the sequence depicted in when optimally d. In yet another embodiment, the fusionprotein comprises at least another XTEN linked to said factor VIII polypeptide at the C-terminus of saidfactor VIII polypeptideor within or optionally replacing the B domain of said factor VIII polypeptide. Ina specific embodiment, the fusion protein comprises at least one XTEN sequence located within oroptionally replacing the B domain of said factor VIII polypeptide. In another specific embodiment, thefusion n comprises at least one XTEN sequence linked to said factor VIII polypeptide at the C-terminus of said factor VIII polypeptide. In one ment, the recombinant factor VIII fusion proteincomprises a B-domain deleted variant of human factor VIII, wherein the B-domain deletion starts from afirst position at about amino acid residue number 741 to about 750 and ending at a second position atamino acid e number 1635 to about 1648 with reference to full-length human factor VIII sequenceas set forth in In another embodiment, the recombinant factor VIII fusion protein comprises afirst XTEN sequence linked to said factor VIII polypeptide at the C-terminus of said factor VIIIpolypeptide, and at least a second XTEN within or replacing the B domain of said factor VIIIpolypeptide, wherein the second XTEN is linked to the C-terminal end of about amino acid residuenumber 741 to about 750 and to the N—terminal end of amino acid residue numbers 1635 to about 1648with reference to full-length human factor VIII sequence as set forth in wherein the cumulativelength of the XTEN is at least about 100 amino acid residues. In one ment, in the ing fusionprotein, the second XTEN links the factor VIII amino acids between N745 to P1640 or between S743 toQ1638 or between P747 to V1642 or between N745 and Q1656 or between N745 and S1657 or betweenN745 and T1667 or n N745 and Q1686 or between R747 and V1642 or between T751 and T1667.
In one embodiment, the recombinant factor VIII fusion protein comprises a sequence haVing at leastabout 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99%, to about 100% sequence identity compared to asequence of comparable length selected from Table 21, when optimally d. In another embodiment,the recombinant factor VIII fusion protein ses at least a second XTEN, optionally a third XTEN,optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, wherein each of thesecond, third, fourth, fifth, or sixth XTEN is linked to said factor VIII polypeptide at a second, third,fourth, fifth, or sixth site selected from the group ting of an insertion site from Table 5, Table 6,Table 7 Table 8, and Table 9; a location within 6 amino acids of amino acid residue 32, 220, 224, 336,339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 ofmature factor VIII; a location between anytwo adjacent domains of said factor VIII polypeptide, wherein said two nt domains are selectedfrom the group consisting ofA1 and A2 domains, A2 and B domains, B and A3 s, A3 and C1domains, and C1 and C2 domains; a location within the B domain of said factor VIII ptide,wherein the second XTEN is linked to the C-terminal end of about amino acid residue number 741 toabout 750 and to the N—terminal end of amino acid residue numbers 1635 to about 1648 of a native factorVIII sequence; and the C-terminus of said factor VIII polypeptide. In one embodiment, the first XTEN isseparated from the second XTEN by at least 10 amino acids, at least 50 amino acids, at least 100 aminoacids, at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids. In one embodimentof the recombinant factor VIII fusion protein that comprises at least a second XTEN, ally a thirdXTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, each XTEN hasat least about 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%,or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about97%, or at least about 98%, or at least about 99%, or about 100% sequence identity compared to anXTEN of comparable length selected from the group consisting of the sequences in Table 4, Table 13,Table 14, Table 15, Table 16, and Table 17, when optimally aligned. In yet another embodiment of therecombinant factor VIII fusion protein that comprises at least a second XTEN, optionally a third XTEN,optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, In preferredembodiments, the recombinant factor VIII fusion protein exhibits a terminal half-life at least about 3hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours,or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administeredto a subject, wherein said subject is selected from human and factor on Willebrand factor doubleknock-out mouse. Further, in the embodiments of this paragraph, the fusion protein exhibits reducedbinding to anti-factor VIII dy or r ed procoagulant activity, or both as compared to acorresponding factor VIII not linked to XTEN. In one embodiment, the procoagulant ty of therecombinant factor VIII fusion protein is at least 30%, or 40%, 50%, 80%, 100%, 200%, 300%, 400%, or500% greater procoagulant actiVity in the presence of the anti-FVIII antibody compared to acorresponding factor VIII not linked to XTEN when each are d by an in Vitro coagulation assay. Inone embodiment, the reduced g of the fusion n to anti-factor VIII antibody is determinedusing a Bethesda assay using anti-factor VIII antibody selected from the group ting of theantibodies of Table 10 and polyclonal antibody from a hemophilia A patient with factor VIII inhibitors,n the d binding and retained procoagulant actiVity of the fusion protein is eVidenced by alower da titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200Bethesda units for the fusion protein ed to that for the factor VIII not linked to XTEN.
In one embodiment, the recombinant factor VIII fusion protein can, for example, comprise oneor more XTEN wherein the XTEN has at least about 80% sequence identity, or about 90%, or about91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, or about 99%, to about 100% sequence identity compared to one or more XTEN of comparablelength selected from Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimallyaligned.
In another aspect, the invention relates to recombinant factor VIII fusion ns comprisingFVIII and one or more XTEN in specific N— to C-terminus configurations. In one embodiment of theCFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula I:(XTEN)X-CF-(XTEN)y Iwherein independently for each occurrence, CF is a factor VIII as defined herein, including sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced fromTable 1; X is either 0 or 1 and y is either 0 or 1 wherein x+y 31; and XTEN is an extended recombinantpolypeptide as described herein, including, but not limited to sequences having at least about 80%, or atleast about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%,or at least about 99% or 100% sequence identity to sequences set forth in Table 4. Accordingly, theCFXTEN fusion composition can have XTEN-CF, XTEN-CF-XTEN, or CF-XTEN configurations.
In r embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula II:(XTEN)X-(S)X-(CF)-(XTEN) y IIwherein independently for each ence, CF is a factor VIII as defined herein, including sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forthin Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionallyinclude a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y iseither 0 or 1 n x+y 21; and XTEN is an extended recombinant polypeptide as described hereinincluding, but not limited to sequences having at least about 80%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100%sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the ion provides a recombinantfactor VIII fusion protein, wherein the fusion protein is of formula III:(XTEN)X-(S)X-(CF)-(S)y-(XTEN)y 111wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequence set for inTable 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can allyinclude a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y iseither 0 or 1 wherein x+y 21; and XTEN is an extended recombinant polypeptide as described hereining, but not limited to sequences having at least about 80%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100%sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a inantfactor VIII fusion protein of formula IV:XTEN)u-(A2)-(XTEN)V-(B)-(XTEN)W-(A3)-(XTEN)x-(C1)-(XTEN)y-(C2)-(XTEN)Zwherein ndently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of theB domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; v is either 0 or 1; w is either 0 or 1;X is either 0 or 1; y is either 0 or 1; y is either 0 or 1 with the proviso that u + v + X + y+z 31; and XTENis an extended recombinant polypeptide as described herein including, but not d to sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forthin Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of a V:(XTEN)t-(S)a -(A1)-(S)b-(XTEN)u-(S)b-(A2)-(S)c-(XTEN)V-(S)c-(B)-(S)d-(XTEN)w-(S)d-(A3)—(S)e-(XTEN)x-(S)e-(C1)-(s)r(XTEN)y-(S)r(cz)-(S)g-(XTEN)Z Vwherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of theB domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence havingbetween 1 to about 50 amino acid residues that can optionally include a cleavage ce or aminoacids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0WO 22617 or 1; W is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t + u + V + w+X + y + z 31; and XTEN is an extended recombinant polypeptide as described herein including, but notlimited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identityto sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine ora sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula VI:(XTEN)u-(S)a-(A1)-(S)b-(XTEN)v-(S)b-(A2)-(S)c-(XTEN)w-(S)c-(A3)-(S)d-(XTEN)x-(S)d-(C1)-(s)e-(XTEN)y-(8)6-(C2)—(S)r(XTEN)Z VIwherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacerce having between 1 to about 50 amino acid residues that can ally include a cleavagesequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either0 or 1; dis either 0 or 1; e is either 0 or 1; fis either 0 or 1; uis either 0 or 1; Vis either 0 or 1; Wis 0 or1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u + V + w+ X + y + z 31; andXTEN is an ed recombinant ptide as described herein including, but not limited tosequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%,or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity tosequences set forth in Table 4. In r embodiment of formula V, the spacer sequence is glycine or asequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula VII:(SP)-(XTEN)x-(CS)X-(S)x-(FVIII_1-745)-(S)y-(XTEN)y-(S)y-(FVIII_1640-2332)-(S)Z-(CS)Z-(XTEN)Z VIIwherein independently for each occurrence, SP is a signal peptide, ably with sequenceMQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is a cleavage ce listed in Table 12, S is aspacer sequence having between 1 to about 50 amino acid residues that can ally include aminoacids compatible with restrictions sites, “FVIII_1-745” is residues 1-745 of Factor FVIII and“FVIII_1640-2332” is residues 1640-2332 of FVIII, X is either 0 or 1, y is either 0 or 1, and z is either 0or 1, wherein x+y+z >2; and XTEN is an eXtended inant polypeptide as described hereining, but not limited to sequences having at least about 80%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100%sequence identity sequences set forth in Table 4. In one embodiment of formula VII, the spacer sequenceis GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine ora sequence selected from Tables 11 and 12.
In r embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula VIII:(A1)—(S)a-(XTEN)V-(S)a-(A2)-(B1)—(S)b-(XTEN)w-(S)b-(B2)-(A3)—(S)c-(XTEN)x-(S)c-(C1)—(S)d-(XTEN)y-(S)d-(C2)-(S)e-(XTEN)Z VIIIwherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;B1 is a fragment of the B domain that can have from residue 741 to 743-750 of FVIII or alternativelyfrom about residue 741 to about residues 745 of FVIII; B2 is a fragment of the B domain that can havefrom residues 1635-1686 to 1689 of FVIII or alternatively from about e 1640 to about residues1689 of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; Sis a spacer sequence having between 1 to about 50 amino acid residues that can optionally include acleavage sequence or amino acids compatible with ctions sites; a is either 0 or 1; b is either 0 or 1; cis either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; V is either 0 or 1; Wis 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 With the proviso that u + V + w+ X + y + z21; and XTEN is an extended recombinant polypeptide as bed herein including, but not limited tosequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%,or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity tosequences set forth in Table 4. In one embodiment of formula VIII, the spacer sequence is GPEGPS(SEQ ID NO: 1612). In another embodiment of a V, the spacer sequence is e or a sequenceed from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula IX:(A1N)'(S)a'(XTEN)I'(S)b'(A1C)'(A2 N)-(S)c-(XTEN)u-(S)d-(A2c)-(BN)-(S)e-(XTEN)v-(S)r(Bc)-(A3N)-(S)g'(XTEN)W'(S)h'(A3C)'(C1N)'(S)i'(XTEN)X'(S)j'(C1C)'(C2N)'(S)k'(XTEN)y'(S)1'(C2C)'(S)m'(XTEN)zWherein independently for each occurrence, AlN is a fragment of the A1 domain from at least residuenumber 1 (numbered relative to , mature FVIII) to no more than residue number 371, A1C is afragment of the A1 domain from at least residue number 2 to no more than residue number 372, With thepriViso that no sequence of the A1N fragment is duplicated in the A1C is a fragment; A2N is a fragment ofthe A2 domain from at least residue number 373 to no more than residue number 739, Me is a fragmentof the A2 domain from at least residue number 374 to no more than residue number 740, With the priVisothat no sequence of the A2N fragment is duplicated in the Me is a fragment; BN is a nt of the Bdomain from at least residue number 741 to no more than residue number 1647, BC is a fragment of the Bdomain from at least residue number 742 to no more than residue number 1648, With the priViso that nosequence of the BN fragment is duplicated in the BC is a fragment; A3N is a fragment of the A3 domainfrom at least residue number 1649 to no more than residue number 2019, A3C is a nt of the A3domain from at least residue number 1650 to no more than e number 2019, With the priViso that nosequence of the A3N fragment is duplicated in the A3C is a fragment; ClN is a fragment of the C1 domainfrom at least residue number 2020 to no more than residue number 2171, C1C is a nt of the C1domain from at least residue number 2021 to no more than residue number 2172, With the o that nosequence of the ClN fragment is duplicated in the C1C is a fragment; C2N is a fragment of the C2 domainfrom at least residue number 2173 to no more than residue number 2331, C2C is a fragment of the C2domain from at least e number 2174 to no more than residue number 2332, with the priviso that nosequence of the C2N fragment is duplicated in the C2C is a fragment; S is a spacer sequence havingbetween 1 to about 50 amino acid residues that can optionally include a cleavage sequence or aminoacids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0or 1; k is either 0 or 1; l is either 0 or 1; m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; V is either 0or 1; W is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t + u + V + w+X + y + z 31; and XTEN is an ed recombinant polypeptide as bed herein including, but notlimited to sequences having at least about 80% sequence identity, or about 90%, or about 91%, or about92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%, to about 100% sequence identity compared to one or more XTEN of comparable length selectedfrom Table 4. In one embodiment of formula IX, the spacer sequence is GPEGPS (SEQ ID NO: 1612).
In another embodiment of formula V, the spacer sequence is glycine or a ce selected from Tables11 and 12. In another embodiment of a IX, Z is 1. In another embodiment of the fusion protein offormula IX V is 1 and the XTEN is linked to the C-terminal end of about amino acid residue number 741to about 750 and to the N—terminal end of amino acid residue numbers 1635 to about 1648 with referenceto full-length human factor VIII sequence as set forth in In another embodiment of the fusionprotein of formula IX, the sum of t, u, v, W, X, y, and 2 equals 2, 3, 4, 5, or 6. In another embodiment offormula IX, the sum of t, u, v, W, X, y, and 2 equals 2, and v is 1 and z is 1. In another embodiment of thefusion protein of formula IX, the sum of t, u, v, W, X, y, and 2 equals 3, v and 2 each equal 1, and either t,u, W, X or y is 1. In another embodiment of a IX, the sum of t, u, v, W, X, y, and 2 equals 4, v andW and 2 each equal 1, and two of t, u, X or y is 1. In another ment of the fusion protein of aIX, the cumulative length of the XTENs is between about 84 to about 3000 amino acid es. Inanother embodiment of formula IX, at least one XTEN is ed immediately downstream of an aminoacid which corresponds to an amino acid in mature native human factor VIII selected from the groupconsisting of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905and 1910. In another embodiment of the fusion protein a IX, each XTEN is linked to said fusionprotein at sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In another embodiment ofthe fusion protein formula IX, each XTEN has at least about 80%, or about 90%, or at least about 91%,or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identitycompared to an XTEN of comparable length selected from the group consisting of the sequences in Table4, Table 13, Table 14, Table 15, Table 16, and Table 17, when lly aligned.
In another embodiment of the CFXTEN composition, the invention provides a firstrecombinant factor VIII polypeptide of formula X:(A1)—a1—(A2)—a2—[B] Xand a second polypeptide comprising Formula XI:a3 — (A3) — (Cl) - (C2) XIwherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, Alis an Al domain of factor VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of factor VIII, afragment thereof, or is deleted; A3 is an A3 domain of factor VIII; Cl is a Cl domain of factor VIII; C2is a C2 domain of factor VIII; al, a2, and a3 are acidic spacer regions; wherein the Al domain comprisesan XTEN permissive loop-l (Al -1) region and an XTEN permissive loop-2 (Al -2) region; wherein theA2 domain comprises an XTEN permissive loop-l (A2-l) region and an XTEN permissive loop-2 (A2-2) ; n the A3 domain comprises an XTEN permissive loop-l (A3-l) region and an XTENpermissive loop-2 (A3 -2) region; wherein an XTEN sequence is inserted into at least one of the regionsAl -1, Al -2, A2-l, A2-2, A3-l, or A3 -2; and wherein the recombinant factor VIII protein exhibitsprocoagulant activity. In one embodiment of the heterodimer, the first polypeptide and the secondpolypeptide form a single polypeptide chain comprising the formula (Al) al (A2) a2 [B] [a3](A3) — (C1) — (C2). In one embodiment of the foregoing, “fused” means a peptidic bond.
In another ment of the CFXTEN composition, the invention provides a firstrecombinant factor VIII polypeptide of formula X:(Al)—al—(A2)—a2—[B] Xand a second polypeptide comprising Formula XI:a3 — (A3) — (Cl) - (C2) XIwherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, Alis an Al domain of factor VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of factor VIII, ant thereof, or is deleted; A3 is an A3 domain of factor VIII; Cl is a Cl domain of factor VIII; C2is a C2 domain of factor VIII; al, a2, and a3 are acidic spacer regions; wherein an XTEN sequence ised into a3; and wherein the recombinant factor VIII protein exhibits procoagulant actiVity. In oneembodiment of the heterodimer, the first polypeptide and the second polypeptide form a singlepolypeptide chain comprising the formula (Al) al (A2) a2 [B] [a3] (A3) (C1) (C2). In oneembodiment of the foregoing, “fused” means a peptidic bond. in embodiments of the foregoing ae X and XI polypeptides, the XTEN permissive loopsare contained within surface-exposed, flexible loop structures, and wherein Al-l is located n betastrand 1 and beta strand 2, Al -2 is located between beta strand 11 and beta strand l2, A2-l is locatedn beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33,A3-l is located between beta strand 38 and beta strand 39 and A3-2 is located between beta strand 45 andbeta strand 46, according to the secondary structure of mature factor VIII stored as Accession Number2R7E of the DSSP database. In other embodiments of the foregoing formulae X and \I ptides, thesurface-exposed, flexible loop structure sing Al-l corresponds to a region in native mature humanfactor VIII from about amino acid 15 to about amino acid 45. In other embodiments ofthe foregoingformulae X and Xi polypeptides the Al -1 corresponds to a region in native mature human factor VIIIfrom about amino acid 18 to about amino acid 41. In other ments of the foregoing formulae X andXI polypeptides, the surface-exposed, flexible loop structure sing A1-2 corresponds to a region innative mature human factor VIII from about amino acid 201 to about amino acid 232. In otherembodiments of the foregoing formulae X and XI polypeptides the A1-2 corresponds to a region innative mature human factor VIII from about amino acid 218 to about amino acid 229. In otherembodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loopstructure comprising A2-1 corresponds to a region in native mature human factor VIII from about aminoacid 395 to about amino acid 421. In other embodiments of the foregoing formulae X and XIpolypeptides, the A2-1 ponds to a region in native mature human factor VIII from about aminoacid 397 to about amino acid 418. In other embodiments of the foregoing formulae X and XIpolypeptides, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region innative mature human factor VIII from about amino acid 577 to about amino acid 635. In otherembodiments of the foregoing formulae X and XI polypeptides, the A2-2 corresponds to a region innative mature human factor VIII from about amino acid 595 to about amino acid 607. In otherembodiments of the foregoing formulae X and XI polypeptides, the e-exposed, flexible loopstructure comprising A3-1 ponds to a region in native mature human factor VIII from about aminoacid 1705 to about amino acid 1732. In other embodiments of the foregoing formulae X and XIpolypeptides, the A3-1 corresponds to a region in native mature human factor VIII from about aminoacid 1711 to about amino acid 1725. In other embodiments of the foregoing formulae X and XIpolypeptides, the the surface-exposed, flexible loop structure sing A3-2 corresponds to a region innative mature human factor VIII from about amino acid 1884 to about amino acid 1917. In otherembodiments of the foregoing formulae X and XI polypeptides, the A3-2 corresponds to a region innative mature human factor VIII from about amino acid 1899 to about amino acid 1911. in otherembodiments of the foregoing formulae X and XI polypeptides, an XTEN sequence is inserted into atleast two of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2. In other embodiments of the ‘oregoin0'0.formulae X and XI polypeptides, an XTEN sequence is inserted immediately downstream of an aminoacid which corresponds to an amino acid in mature native human factor VIII selected from the groupconsisting of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905and 1910. In other embodiments of the foregoing ae X and Xl polypeptides, an additional XTENce is inserted into the a3 acidic spacer region. In other embodiments of the foregoing ae Xand XI polypeptides, an additional XTEN ce is inserted into the a3 acide spacer atelydownstream of an amino acid which corresponds to amino acid 1656. In other embodiments of theforegoing formulae X and XI polypeptides, the A1 domain comprises an XTEN permissive loop-1 (A1-1) region and an XTEN permissive loop-2 (A1-2) region wherein the A2 domain comprises an XTENpermissive loop-1 (A2-1) region and an XTEN permissive loop-2 (A2-2) region, and wherein the A3domain comprises an XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2 (A3 -2)region, and wherein an additional XTEN sequence is inserted into at least one of the regions A1-1, A1-2,A2-1, A2-2, A3-1, or A3 -2. In other embodiments of the foregoing formulae X and XI polypeptides, anonal XTEN sequence is inserted immediately downstream of an amino acid which ponds toan amino acid in mature native human factor VIII ed from the group consisting of amino acidresidue number 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In theforegoing embodiments of ae X and Xi polypeptides, the fusion protein ts at least about%, 40%, 50%, 60%, 70%, or 80%, or 90% of the procoagulant activity of the corresponding factorVIII not linked to XTEN, wherein the procoagulant activity is assayed by an in vitro coagulation assay.
In all ments, the polypeptide can, for example, exhibit an in vitro procoagulant activityexceeding 0.5 IU/ml, or 1.0, or 1.5, or 2.0 IU/ml When expressed in cell-culture medium and assayed byan in vitro coagulation assay. The procoagulant activity can be measured by a chromogenic assay, a onestage clotting assay (e. g., a aPTT) or both.
In some ments, Wherein the inant factor VIII fusion protein comprises a factorVIII and at least a first and a second XTEN, the at least first XTEN is separated from the at least secondXTEN by at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 aminoacids, at least 300 amino acids, or at least 400 amino acids.$026} In preferred embodiments, the recombinant factor VIII fusion protein comprising a factor VIIIand at least a first XTEN and, optionally, at least a second, or optionally at least a third, or ally atleast a fourth XTEN, the fusion protein exhibits reduced binding to an actor VIII antibody ascompared to the corresponding factor VIII not linked to XTEN. The reduced binding can be assessedeither in vivo or by an in vitro assay. In one embodiment, the in vitro assay is an ELISA assay, Whereinthe binding of an anti-FVIII antibody to the fusion n is reduced at least about 5%, 10%, 15%, 20%,%, 30%, 35% or at least about 40% or more compared to a FVIII not linked to XTEN. In anotherembodiment, the in vitro assay is a Bethesda assay Wherein the reduced binding of the fusion protein isevidenced by a lower Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100,or 200 Bethesda units for the fusion n compared to that for a factor VIII not linked to XTEN. Inthe in vitro assays, the anti-factor VIII antibody is selected from an antibody of Table 10 and polyclonalantibody from a hemophilia A patient With factor VIII inhibitors. In particular ments of arecombinant factor VIII fusion protein sing a factor VIII and at least a first and a second XTENting reduced binding to a factor VIII inhibitor antibody, the first XTEN is linked to said factor VIIIpolypeptide Within a C2 domain of said factor VIII polypeptide, and the second XTEN is linked to saidfactor VIII polypeptide Within an A1 or A2 domain of said factor VIII polypeptide, Wherein said fusionprotein ts reduced binding to a factor VIII inhibitor antibody as compared to the correspondingfactor VIII not linked to XTEN, Wherein the factor VIII inhibitor antibody is capable of binding to anepitope located Within the A1, A2 or C2 domain, and further n the fusion protein exhibitsprocoagulant activity. In one embodiment of the foregoing fusion protein, the second XTEN is linked tosaid factor VIII polypeptide with in the A2. domain of the factor VIII polypeptide and the factor VIIIinhibitor antibody binds to the A2 domain of the factor VIII polypeptide. In another ment of theforegoing fusion protein, the second XTEN is linked to said factor VIII polypeptide Within the (.2domain of the factor VIII polypeptide and the factor VIII inhibitor antibody binds to the C2 domain ofthe factor VIII polypeptide. The binding of an anti—factor VIII antibody to the fusion protein is reducedby at least about 5%, I094), I596, 20%, 25%, 30%, 35% or 40% compared to the eorreoponding factor 2012/046326VIII not ed to XTEN when assayed by an ELISA assay, wherein the anti~factor \HII antibody isselected from the group consisting of the antibodies in Table l0 and a polyclonal antibody from ahemophilia A subject with factor Vlll inhibitors. The foregoing fusion proteins can further comprise atleast three XTEN s, wherein the at least third XTEN is linked to the factor VIII at a site ed fromwithin or replacing the B domain, at the {TL-terminus, and at or Within 1, 2, 3, 4, 5, or 6 amino acids of aninsertion site selected from Table 7 or Table 9. in the ments with d binding to anti-factorVlll antibodies, the fusion protein has greater procoagulant activity in the presence of the anti—FVlllantibody of at least l0%, 20%, 30%, 40%., 50%, 80%, l00%, 200%, 300%, 400%, or 500%. or morecompared to a corresponding factor VIII not linked to XTEN when assayed by an in Vll't’O ationassay (eg, a chromogenic or one—stage clotting assay).{0027} in all embodiments, the XTEN of the fusion protein can, for example, be characterized in thatthe XTEN comprise at least 36, or at least 42, or at least 72, or at least 96, or at least 144, or at least 288,or at least 400, or at least 500, or at least 576, or at least 600, or at least 700, or at least 800, or at least864, or at least 900, or at least 1000, or at least 2000, to about 3000 amino acid es or even moreresidues; the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P)residues constitutes at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%,or at least about 97%, or at least about 98%, or at least about 99% of the total amino acid residues of theXTEN; the XTEN is substantially non-repetitive such that (i) the XTEN contains no three uousamino acids that are identical unless the amino acids are serine; (ii) at least about 80% of the XTENsequence consists of erlapping sequence motifs, each of the ce motifs comprising about 9to about 14, or about 12 amino acid residues consisting of four to six amino acids selected from glycine(G), alanine (A), serine (S), threonine (T), ate (E) and proline (P), wherein any two contiguousamino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii)the XTEN ce has a subsequence score of less than 10; the XTEN has greater than 90%, or greaterthan 95%, or greater than 99% random coil formation as determined by GOR algorithm; the XTEN hasless than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm; the XTENlacks a predicted T-cell epitope When analyzed by TEPITOPE algorithm, wherein the PEthreshold score for said prediction by said algorithm has a threshold of —9, and wherein said fusionprotein exhibits a terminal half-life that is longer than at least about 12 h, or at least about 24 h, or at leastabout 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, orat least about 21 days or greater. In one embodiment, the recombinant factor VIII fusion proteincomprises at least a second, or at least a third, or at least a fourth XTEN, Which can be identical ordifferent to the other XTEN. According to a different approach, the at least one, at least a second, or atleast a third, or at least a fourth XTEN of the CFXTEN fusion protein each have at least about 80%ce identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identitycompared to one or more XTEN of comparable length selected from Table 4, Table 13, Table 14, Table, Table 16, and Table 17, When optimally aligned. In yet another different approach, the at least one, atleast a second, or at least a third, or at least a fourth XTEN of the CFXTEN fusion protein each have atleast 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequenceselected from AE42_1, AE42_2, AE42_3, AG42_1, AG42_2, AG42_3, AG42_4, AE144_1A,AE144_2A, AE144_2B, AE144_3A, 3B, AE144_4A, AE144_4B, 5A, AE144_6B,AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, F, AG144_3, AG144_4, AE288_1,AE288_2, AG288_1, and AG288_2.{0028} In one embodiment, the factor ‘v’lll component of the CFXTEN recombinant factor Vlll fusionprotein eon’iprisies one, two or three amino acid substitutions selected from es R1648, Yl680, andR1689, numbered relative to mature human factor VIII, wherein the substitutions are selected fromalanine, glycine, and phenylalanine. Non-limiting es ofsaid substitutions e Rl648A,YléSQE, and R1689A.{002,9} ln another embodiment, the CFXTEN fusion protein exhibits an apparent molecular weightlactor of at least about L3, or at least about two, or at least about three, or at least about four, or at leastabout five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or atleast about 10, when measured by size exclusion tography or comparable method.
In some embodiments of the CFXTEN fusion proteins, one or more of the XTEN is to theFVIII Via one or two cleavage sequences that each is cleavable by a mammalian protease selected fromthe group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor Ila(thrombin), se-2, MMP-12, MMP13, MMP-17 and MMP-20, wherein cleavage at the cleavagesequence by the mammalian protease es the factor VIII ce from the XTEN sequence, andwherein the released factor VIII sequence exhibits an increase in procoagulant activity compared to theved fusion protein. In one embodiment, the cleavage sequence(s) are cleavable by factor XIa.
According to a different approach, the CFXTEN fusion proteins comprise at least three XTENslocated at different locations of the factor VIII polypeptide, wherein said different locations are selectedfrom: an insertion location at or Within 1 to 6 amino acids from a site selected from Table 5, Table 6,Table 7 Table 8, and Table 9; a location at or Within 1 to 6 amino acids of amino acid residue 32, 220,224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 ofmature factor VIII; a locationbetween any two adjacent domains in the factor VIII ce, n said two adjacent domains areselected from the group consisting of A1 and A2, A2 and B, B and A3, A3 and C1, and C1 and C2; alocation Within an internal B domain deletion starting from a first position at about amino acid residuenumber 741 to about 750 and ending at a second position at amino acid residue number 1635 to about1648 With reference to full-length human factor VIII sequence as set forth in and the C-terminusof the factor VIII sequence, n the cumulative length of the multiple XTENs is at least about 100 toabout 3000 amino acid residues and wherein the fusion protein retains at least about 30%, or about 40%,or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activitycompared to the corresponding factor VIII not linked to XTEN, wherein the procoagulant activity isassayed by an in Vitro coagulation assay. In one embodiment of the foregoing, the fusion protein exhibitsa prolonged terminal half-life when administered to a subject as compared to a corresponding factor VIIIpolypeptide lacking said XTEN, wherein said fusion protein ts a terminal half-life at least about 3hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours,or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administeredto a subject. In one embodiment, the subject is selected from the group consisting of human and a factorVIII/von Willebrand factor double knock-out mouse. In one embodiment of the foregoing, the fusionprotein does not se a sequence selected from TASSSP (SEQ ID NO: 31),GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ IDNO: 34), andGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing, the fusion n does notcontain an XTEN ce consisting ofGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59),PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS (SEQ ID NO: 71), orPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS (SEQ ID NO: 80).
In a timber aspect, the invention concerns CFXTEN fusion proteins with enhancedpharmacokinetic properties, including enhanced parameters compared to FVIII not linked to XTEN,wherein the enhanced properties e but are not limited to longer terminal half-life, larger area underthe curve, increased time in which the blood concentration remains within the therapeutic window,increased time between consecutive doses results in blood concentrations within the therapeutic window,and decreased dose in IU over time that can be administered compared to a FVIII not linked to XTEN,yet still result in a blood concentration above a threshold concentration needed for a procoagulant effect.
In some embodiments, a CFXTEN fusion proteins exhibit a prolonged al half-life whenstered to a subject as compared to a corresponding factor VIII polypeptide lacking said XTEN.
The subject can be a human or a mouse, such as a factor VIII/von Willebrand factor double outmouse. In one embodiment of the foregoing, the CFXTEN exhibits a terminal half-life that is at leastabout two-fold, or about three fold, or about four-fold, or about five-fold, or about 10-fold, or about 20-fold longer when administered to a subject compared to the corresponding factor VIII not linked toXTEN. In one embodiment, the CFXTEN fusion protein exhibits a al half-life at least about 3hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours,or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when steredto the subject. In other embodiments, the ed pharmacokinetic property of the fiJsion proteins ofthe embodiments is the property of maintaining a circulating blood concentration of procoagulant fusionprotein in a subject in need thereof above a threshold concentration of 0.01 IU/ml, or 0.05 IU/ml, or 0.1IU/ml, or 0.2 IU/ml, or 0.3 IU/ml, or 0.4 IU/ml or 0.5 IU/ml for a period that is at least about two fold, orat least about three-fold, or at least about old, or at least about five-fold, or at least about six-fold,or at least about eight-fold, or at least about ten-fold, or at least about 20-fold, or at least about 40-fold, orat least about d longer compared to the corresponding FVIII not linked to XTEN and administeredto a subject at a comparable dose. The increase in half-life and time spent above the thresholdconcentration permits less frequent dosing and decreased amounts of the fusion protein (in molesequivalent) that are stered to a subject, compared to the corresponding FVIII not linked to XTEN.
In one embodiment, stration of a subject fusion protein to a subject using a therapeutically-effective dose regimen results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold, or at least six-fold, or at least eight-fold, or at least 10-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold or higher between at least two consecutive CmaXpeaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding FVIIInot linked to the XTEN and administered using a comparable dose regimen to a t.
In preferred embodiments, the CFXTEN fusion proteins retain at least about 30%, or about40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulantactivity compared to the ponding factor VIII not linked to XTEN, wherein the procoagulantactivity is assayed by an in vitro coagulation assay such as, but not limited to a chromogenic assay or aone- or two-stage clotting assay.
According to a different approach, the invention provides recombinant factor VIII fusionproteins comprising a factor VIII polypeptide and at least one extended recombinant polypeptide(XTEN), n said factor VIII polypeptide comprises A1 , A2 domain, A3 domain, C1domain, C2 domain and ally all or a portion of B domain, and wherein said at least one XTEN islinked to said factor VIII polypeptide at an insertion site selected form residue numbers 18-32, or 40, or211-224, or 3, or 599, or 745-1640, or 1656-1728, or 1796-1804, or 1900-1912, or 2171-2332;and n the fusion protein retains at least about 30%, or about 40%, or about 50%, or about 60%, orabout 70%, or about 80%, or about 90% of the gulant activity compared to the correspondingfactor VIII not linked to XTEN. In one embodiment of the foregoing, the fusion protein comprises atleast a second XTEN, or at least a third, or at least a fourth XTEN n the XTEN are linked to thefactor VIII at a site at or within 1 to 6 amino acids of a site selected from Table 5, Table 6, Table 7, Table8, and Table 9. In another embodiment, the invention provides an recombinant factor VIII fusion proteinfurther comprising at least a second XTEN, or at least a third, or at least a fourth XTEN linked to saidFVIII polypeptide at an insertion site selected from Table 5, Table 6, Table 7, Table 8, Table 9, at orwithin 6 amino acids to the N— or C-terminus side of an insertion location at one or more insertionlocations from Figure 8 and within one or more insertion ranges from Figure 9 wherein at least twoXTEN are separated by an amino acid sequence of at least 100 to about 400 amino acids.
The invention provides CFXTEN wherein the XTEN have a Ratio XTEN Radii of at least 2.3or at least 2.5, and are separated by an amino acid sequence of at least about 20 amino acid residues, or atleast about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400amino acid residues. In other embodiments, the CFXTEN comprise at least four XTEN wherein theXTEN have a Ratio XTEN Radii of at least 2.3, or at least 2.5, or at least 2.8, and n at least threeof the four of the XTEN linked to the fusion protein are separated by an amino acid sequence of at leastabout 20 amino acid es, or at least about 50, or at least about 100, or at least about 200, or at leastabout 300, or at least about 400 amino acid residues, and the fourth XTEN is linked within the B domain(or a fragment thereof) or within the C domain (or the terminus thereof).
In some ments, the subject compositions are configured to have reduced binding affinityfor a nce receptor in a t as compared to the corresponding FVIII not linked to the XTEN. Inone embodiment, the CFXTEN fusion protein exhibits g affinity for a clearance receptor of theFVIII in the range of about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or about2% to about 10% of the binding affinity of the corresponding FVIII not linked to the XTEN. In rembodiment, a fusion protein with reduced affinity for a clearance receptor has reduced active clearanceand a corresponding increase in half-life of at least about 2-fold, or 3-fold, or at least 4-fold, or at leastabout 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold,or at least about 9-fold, or at least about 10-fold, or at least about 12-fold, or at least about 15 -fold, or at least about 17-fold,or at least about 20-fold longer compared to the corresponding FVIII that is not linked to the XTEN.
In an embodiment, the invention provides a recombinant factor VIII fusion protein singFVIII and one or more XTEN wherein the fusion protein exhibits increased solubility of at least three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least aboutfold, or at least about eight-fold, or at least about nine-fold, or at least about ld, or at leastabout 15-fold, or at least a 20-fold, or at least 40-fold, or at least 60-fold at physiologic conditionscompared to the FVIII not linked to XTEN.
In a further aspect, the invention provides a pharmaceutical composition comprising the fusionprotein of any of the embodiments described herein and a pharmaceutically acceptable carrier.
In another embodiment, the invention es a method of treating a coagulopathy in asubject, comprising administering to said subject a composition comprising a clotting effective amount ofthe pharmaceutical ition. In one ment of the method, after said administration, a bloodconcentration of procoagulant factor VIII is maintained at about 0.05, or 1, or 1.5 IU/ml or more for atleast 48 hours after said administration. In another embodiment, the invention provides a method ofclotting blood in a subject, comprising contacting a clotting effective amount of the pharmaceuticalcomposition with the blood.
In another embodiment, the invention provides a method of treating a opathy in a subjectwith circulating inhibitors of factor VIII, comprising administering to said subject a compositioncomprising a therapeutically effective amount of the pharmaceutical ition of , whereinthe composition ts greater procoagulant activity in said subject compared to a compositioncomprising the corresponding factor VIII not linked to XTEN and administered using a ableamount. In one ment of the method, the coagulopathy is hemophilia A. In another embodiment,the coagulopathy is the result of trauma or surgery or infection.
The invention provides a method of treating a bleeding episode in a subject, comprisingadministering to said t a composition comprising a clotting effective amount of the CFXTENpharmaceutical composition, wherein the clotting effective amount of the fusion protein arrests ableeding episode for a period that is at least three-fold, or at least four-fold, or at least five-fold longercompared to a corresponding factor VIII not linked to XTEN and administered using a comparableamount to said subject. miting es of a ponsing factor VIII not linked to XTENinclude native FVIII, the sequences of Table l, BDD-FVIII, and the pCB0114 FVIII.
In another embodiment, the invention provides a CFXTEN recombinant factor VIII fusionprotein for use in a pharmaceutical regimen for treating a hemophilia A patient, said regimen comprisinga pharmaceutical composition sing a CFXTEN fusion n. In one embodiment of thepharmaceutical regimen, the regimen further comprises the step of determining the amount ofceutical composition comprising the CFXTEN needed to achieve hemostasis in the hemophilia Apatient. In another embodiment, the pharmaceutical n for treating a hemophilia A subjectcomprises administering the pharmaceutical composition in two or more successive doses to the subjectat an effective amount, wherein the administration results in at least a 10%, or 20%, or 30%, or 40%, or50%, or 60%, or 70%, or 80%, or 90% r improvement of at least one, two, or three parametersassociated with the hemophilia A disease compared to the factor VIII not linked to XTEN andadministered using a comparable dose. Non-limited examples of parameters improved include bloodconcentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, areduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reducedchromogenic assay time, a reduced bleeding assay time, resolution of a bleeding event, or a dda titer to native FVIII.
In another aspect, the invention provides isolated nucleic acid ces encoding the fusionproteins of any one of the embodiments of the CFXTEN fusion protein. In one ment, the ednucleic acid is the complement of a ce encoding a CFXTEN fusion protein of the embodiments.
In one ment, the isolated nucleic acid further comprises a sequence encoding a signal peptide,wherein said sequence isATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ IDNO: 1613), or the complement thereof In another embodiment, the invention provides an expressionvector comprising the nucleic acid encoding the fusion protein, or the complement thereof. In anotherembodiment, the invention provides an isolated host cell comprising the foregoing expression vector. Inanother embodiment, the invention provides a method of producing the fusion protein of any of theembodiments, comprising providing a host cell comprising the expression vector; culturing the host cellto effect production of the fiision protein; and recovering the fusion protein.
In one embodiment, the invention provides an isolated fusion protein comprising a polypeptidehaving at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%,or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100%sequence identity compared to a sequence of comparable length selected from Table 21, when optimallyaligned.
In another ment, the invention provides an isolated nucleic acid comprising acleotide sequence selected from (a) a ce having at least about 80% sequence identity, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequence ofcomparable length selected from Table 21, when optimally aligned, or (b) the complement of thepolynucleotide of (a). In another embodiment, the isolated c acid comprises the sequenceATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ IDNO: 1613) linked to the 5’ end of the c acid of (a) or the complement of the sequence linked to the3 ’ end of (b).
It is specifically contemplated that the recombinant factor VIII fusion proteins can exhibit oneor more or any combination of the properties disclosed .[0046A] In one aspect, the invention provides a recombinant factor VIII fusion protein comprising afactor VIII polypeptide fused to an extended recombinant polypeptide (XTEN), wherein the factor VIIIpolypeptide comprises A1 domain, A2 domain, A3 domain, a3 domain, C1 domain, C2 domain, andoptionally a B domain or a portion thereof, n the XTEN is inserted within the factor VIIIpolypeptide.[0046B] In one embodiment, the XTEN is ed into the B domain or the portion thereof.[0046C] In r embodiment, the XTEN is inserted into the A1 domain, the A2 , the A3, the 33 , or any combination thereof.[0046B] In a further embodiment, the A1 domain comprises a permissive loop-1 (Al-l) region and asive loop-2 (Al-2) region wherein the XTEN is inserted into Al-l, A1-2, or both.[0046E] In another embodiment, the A2 domain comprises a permissive loop-l (AZ-l) region and apermissive loop-2 (A2—2) region wherein the at least one XTEN is inserted into A2-1, A2-2, or both.[0046F] In a further embodiment, the A3 domain comprises a permissive loop-1 (A3-l) region and apermissive loop—2 (A3 -2) region wherein the at least one XTEN is inserted into A3-1, A3—2, or both.[0046G] In another embodiment, the permissive loop-l (Al-1) region corresponds to a region in nativemature human factor VIII from amino acid 15 to amino acid 45 or from amino acid 18 to amino acid 32of SEQ ID N012, and wherein the XTEN is inserted into Al-l.[0046H] In a further embodiment, the permissive loop-2 (Al—2) region corresponds to a region in nativemature human factor VIII from amino acid 201 to amino acid 232 or from amino acid 211 to amino acid224 of SEQ ID NO: 2, and wherein the XTEN is inserted into Al—2.
In another embodiment, the permissive loop-1 (AZ-1) region corresponds to a region in nativemature human factor VIII from amino acid 395 to amino acid 421 or from amino acid 397 to amino acid418 of SEQ ID NO: 2, and wherein the XTEN is inserted into A2-1.[0046.)] In a further embodiment, the permissive loop-2 (A2—2) region ponds to a region in nativemature human factor VIII from amino acid 577 to amino acid 635 or from amino acid 595 to amino acid607 of SEQ ID NO: 2, and wherein the XTEN is inserted into A2-2.[0046K] In another embodiment, the permissive loop-1 (A3-1) region corresponds to a region in nativemature human factor VIII from amino acid 1705 to amino acid 1732 or from amino acid 1711 to aminoacid 1725 of SEQ ID NO: 2, and wherein the XTEN is inserted into A3-1.[0046L] In a r embodiment, the permissive loop-2 (A3 -2) region corresponds to a region in nativemature human factor VIII from amino acid 1884 to amino acid 1917 or from amino acid 1900 to aminoacid 1912 of SEQ ID NO: 2, and wherein the XTEN is inserted into A3-2.[0046M] In another embodiment, the XTEN is inserted within Al-l immediately downstream of anamino acid ponding to amino acid 17 of SEQ ID NO: 2, amino acid 18 of SEQ ID NO: 2, aminoacid 22 of SEQ ID NO: 2, amino acid 24 of SEQ ID NO: 2, amino acid 26 of SEQ ID NO: 2, amino acid28 of SEQ ID NO: 2, amino acid 32 of SEQ ID NO: 2, amino acid 38 of SEQ ID NO: 2, amino acid 40of SEQ ID NO: 2, or amino acid 41 of SEQ ID NO: 2.[0046N] In a r embodiment, the XTEN is inserted within A1-2 immediately downstream of anamino acid corresponding toamino acid 205 of SEQ ID NO: 2, amino acid 210 of SEQ ID NO: 2, aminoacid 211 of SEQ ID NO: 2, amino acid 216 of SEQ ID NO: 2, amino acid 220 of SEQ ID NO: 2, aminoacid 222 of SEQ ID NO: 2, amino acid 223 of SEQ ID NO: 2, amino acid 224 of SEQ ID NO: 2, oramino acid 230 of SEQ ID NO: 2.
In another embodiment, the XTEN is inserted within A2-1 immediately downstream of anamino acid corresponding to amino acid 399 of SEQ ID NO: 2, amino acid 403 of SEQ ID NO: 2, aminoacid 405 of SEQ ID NO: 2, amino acid 409 of SEQ ID NO: 2, or amino acid 416 of SEQ ID NO: 2.
] In a further embodiment, the XTEN is inserted within A2-2 ately downstream of anamino acid corresponding to amino acid 598 of SEQ ID NO: 2, amino acid 599 of SEQ ID NO: 2, aminoacid 603 of SEQ ID NO: 2, or amino acid 616 of SEQ ID NO: 2.
] In another embodiment, the XTEN is inserted within A3-1 immediately downstream of anamino acid corresponding to amino acid 1711 of SEQ ID NO: 2, amino acid 1713 of SEQ ID NO: 2,amino acid 1720 of SEQ ID NO: 2, amino acid 1724 of SEQ ID NO: 2, amino acid 1725 of SEQ ID NO:2, or amino acid 1726 of SEQ ID NO: 2.[0046R] In a further embodiment, the XTEN is inserted Within A3 -2 immediately downstream of anamino acid corresponding to amino acid 1896 of SEQ ID NO: 2, amino acid 1900 of SEQ ID NO: 2,amino acid 1904 of SEQ ID NO: 2, amino acid 1905 of SEQ ID NO: 2, or amino acid 1910 of SEQ IDNO: 2.
] In another embodiment, the XTEN is inserted within a3 ately downstream of an aminoacid corresponding to amino acid 1656 of SEQ ID NO: 2.[0046T] In a further embodiment, the at the least one XTEN is inserted immediately downstream of anamino acid corresponding to amino acid 740 or 745 of SEQ ID NO: 2.[0046U] In another embodiment, the XTEN is inserted within the C1 or the C2 domain.[0046V] In a further embodiment, the XTEN is inserted immediately downstream of an amino acidcorresponding to an amino acid selected from the group consisting of amino acid 2020 of SEQ ID NO:2, amino acid 2044 of SEQ ID NO: 2, amino acid 2068 of SEQ ID NO: 2, amino acid 2073 of SEQ IDNO: 2, amino acid 2090 of SEQ ID NO: 2, amino acid 2092 of SEQ ID NO: 2, amino acid 2093 of SEQID NO: 2, amino acid 2111 of SEQ ID NO: 2, amino acid 2115 of SEQ ID NO: 2, amino acid 2120 of.
SEQ ID NO: 2, amino acid 2125 of SEQ ID NO: 2, amino acid 2171 of SEQ ID NO: 2, amino acid 2173of SEQ ID NO: 2, amino acid 2188 of SEQ ID NO: 2, amino acid 2223 of SEQ ID NO: 2, amino acid2224 of SEQ ID NO: 2, amino acid 2227 of SEQ ID NO: 2, amino acid 2268 of SEQ ID NO: 2, aminoacid 2277 of SEQ ID NO: 2, amino acid 2278 of SEQ ID NO: 2, or amino acid 2290 of SEQ ID NO: 2.[0046W] In r ment, the XTEN is inserted immediately downstream of an amino acidcorresponding to an amino acid selected from the group consisting of amino acid 18 of SEQ ID NO: 2,amino acid 22 of SEQ ID NO: 2, amino acid 26 of SEQ ID NO: 2, amino acid 28 of SEQ ID NO: 2,amino acid 32 of SEQ ID NO: 2, amino acid 40 of SEQ ID NO: 2, amino acid 211 of SEQ ID NO: 2,amino acid 216 of SEQ ID NO: 2, amino acid 220 of SEQ ID NO: 2, amino acid 224 of SEQ ID NO: 2,amino acid 333 of SEQ ID NO: 2, amino acid 336 of SEQ ID NO: 2, amino acid 339 of SEQ H3 NO: 2,amino acid 399 of SEQ ID NO: 2, amino acid 403 of SEQ ID NO: 2, amino acid 409 of SEQ ID NO: 2,amino acid 416 of SEQ ID NO: 2, amino acid 599 of SEQ ID NO: 2, amino acid 603 of SEQ ID NO: 2,amino acid 740 of SEQ ID NO: 2, amino acid 745 of SEQ ID NO: 2, amino acid 1656 of SEQ ID NO: 2,amino acid 1711 of SEQ ID NO: 2, amino acid 1720 of SEQ ID NO: 2, amino acid 1796 of SEQ ID NO:2, amino acid 1802 of SEQ ID NO: 2, amino acid 1900 of SEQ ID NO: 2, amino acid 1904 of SEQ IDNO: 2, amino acid 1905 of SEQ ID NO: 2, amino acid 1910 of SEQ ID NO: 2, amino acid 1656 of SEQID NO: 2 amino acid 2068 of SEQ ID NO: 2, amino acid 2171 of SEQ ID NO: 2, amino acid 2227 ofSEQ ID NO: 2, or amino acid 2277 of SEQ ID NO: 2.[0046X] In a further embodiment, the recombinant factor VIII fusion protein comprises two XTENs.[0046Y] In another ment, the two XTENs are inserted or fused immediately downstream of oneor two amino acids corresponding to amino acid 18 of SEQ ID NO: 2, amino acid 26 of SEQ ID NO: 2,amino acid 40 of SEQ ID NO: 2, amino acid 399 of SEQ ID NO: 2, amino acid 403 of SEQ ID NO: 2,amino acid 599 of SEQ ID NO: 2, amino acid 745 of SEQ ID NO: 2, amino acid 1656 of SEQ ID NO: 2,amino acid 1711 of SEQ ID NO: 2, amino acid 1720 of SEQ ID NO: 2, amino acid 1725 of SEQ ID NO:2, amino acid 1900 of SEQ ID NO: 2, amino acid 1905 of SEQ ID NO: 2, or amino acid 2332 of SEQID NO: 2.
] In a further embodiment, the two XTENs are inserted or fused immediately downstream of theamino acids corresponding to:i. amino acids 745 and 2332 of SEQ ID NO: 2;ii. amino acids 1656 and 2332 of SEQ ID NO: 2;iii. amino acids 26 and 403 of SEQ ID NO: 2;iv. amino acids 40 and 403 of SEQ ID NO: 2;V. amino acids 18 and 403 of SEQ ID NO: 2;vi. amino acids 26 and 599 of SEQ ID NO: 2;vii. amino acids 40 and 599 of SEQ ID NO: 2;viii. amino acids 18 and 599 of SEQ ID NO: 2;ix. amino acids 18 and 1656 of SEQ ID NO: 2;x. amino acids 26 and 1656 of SEQ ID NO: 2;xi. amino acids 40 and 1656 of SEQ ID NO: 2;xii. amino acids 403 and 1656 of SEQ ID NO: 2;xiii. amino acids 1656 and 1720 of SEQ ID NO: 2;xiv. amino acids 40 and 399 of SEQ ID NO: 2;xv. amino acids 26 and 1900 of SEQ ID NO: 2;xvi. amino acids 18 and 399 of SEQ ID NO: 2;xvii. amino acids 40 and 399 of SEQ ID NO: 2; orxviii. amino acids 1656 and 1900 of SEQ ID NO: 2.[0046AA] In another embodiment, the recombinant factor VIII fusion protein comprises three XTENS,four XTENs, five XTENs, or six XTENs.[0046AB] In a further embodiment:i. the three XTENs are inserted or fused immediately ream of one or more aminoacids corresponding to amino acid 18 of SEQ ID NO: 2, amino acid 26 of SEQ ID NO: 2,amino acid 40 of SEQ ID NO: 2, amino acid 399 of SEQ ID NO: 2, amino acid 403 of SEQID NO: 2, amino acid 599 of SEQ ID NO: 2, amino acid 745 of SEQ ID NO: 2, amino acid1656 of SEQ ID NO: 2, amino acid 1711 of SEQ ID NO: 2, amino acid 1720 of SEQ ID NO:2, amino acid 1725 of SEQ ID NO: 2, amino acid 1900 of SEQ ID NO: 2, amino acid 1905of SEQ ID NO: 2, amino acid 1910 of SEQ ID NO: 2, or amino acid 2332 of SEQ ID NO: 2;ii. the four XTENs are inserted or fused immediately downstream of one or more aminoacids corresponding to amino acid 18 of SEQ ID NO: 2, amino acid 26 of SEQ ID NO: 2,amino acid 40 of SEQ ID NO: 2, amino acid 403 of SEQ ID NO: 2, amino acid 409 of SEQID NO: 2, amino acid 745 of SEQ ID NO: 2, amino acid 1656 of SEQ ID NO: 2, amino acid1720 of SEQ ID NO: 2, amino acid 1900 of SEQ ID NO: 2, amino acid 1905 of SEQ ID NO:2, amino acid 1910 of SEQ ID NO: 2, or amino acid 2332 of SEQ ID NO: 2; oriii. the five XTENs are inserted or fused immediately downstream of one or more aminoacids corresponding to amino acid 18 of SEQ ID NO: 2, amino acid 403 of SEQ ID NO: 2,amino acid 745 of SEQ ID NO: 2, amino acid 1656 of SEQ ID NO: 2, amino acid 1720 ofSEQ ID NO: 2, amino acid 1900 of SEQ ID NO: 2, or amino acid 2332 of SEQ ID NO: 2.[0046AC] In another embodiment, the XTEN comprises 36 amino acids, 42 amino acids, 72 aminoacids, 96 amino acids, 144 amino acids, or 288 amino acids.[0046AD] In a further embodiment, the XTEN comprises 72 amino acids, 144 amino acids, or 288amino acids.[0046AE] In another embodiment, the XTEN comprises one or more XTEN ce motifs, which areselected from the group consisting of SEQ ID N05: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48.[0046AF] In a further embodiment, the one or more XTEN sequence motifs are selected from the groupconsisting of SEQ ID NOS: 23, 24, 25, and 26.[0046AG] In another ment, the XTEN comprises an amino acid sequence having 90%, 96%,97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID N03: 49, 50, 51, 52, 57, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 78, and 79.[0046AH] In a further ment, the XTEN comprises an amino acid sequence having 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,and 351.[0046AI] In another embodiment, the factor VIII polypeptide comprises a native factor VIIIpolypeptide.[0046AJ] In a further embodiment, the factor VIII polypeptide comprises a partially deleted B domainor a fully deleted B domain.[0046AK] In another embodiment, the factor VIII polypeptide comprises a single chain factor VIIIpolypeptide.[0046AL] In a further embodiment, a heterologous polypeptide is fused to the factor VIII polypeptide.[0046AM] In another embodiment, the heterologous polypeptide is an Fc fragment of globulinor an FcRn binding domain.[0046AN] In a further embodiment, the heterologous polypeptide is selected from the group consistingof n or a fragment thereof, a [3 subunit of C-terminal e of human chorionic tropin, aHAP ce, a transferrin, a PAS polypeptide, a polyglycine linker, a polyserine linker, and acombination f.[0046AC] In another aspect, the present invention provides an isolated c acid encoding therecombinant factor VIII fusion protein of the present invention.[0046AP] In a r aspect, the present invention provides a vector comprising the isolated cacid of the present invention.[0046AQ] In one embodiment, the vector is a plasmid, a cosmid, a viral particle, or phage.[0046AR] In another aspect, the present invention provides a host cell sing the vector of thepresent invention.[0046AS] In a further aspect, the present invention provides a pharmaceutical composition comprisingthe recombinant factor VIII fusion protein of the present invention and a pharmaceutically acceptablecarrier.[0046AT] In another , the present invention provides a pharmaceutical composition comprisingthe isolated c acid of the present invention, the vector of the t invention, or the host cell ofthe present invention, and a pharmaceutically able carrier.[0046AU] In a further aspect, the present ion provides a method of making a recombinant factorVIII fusion protein comprising culturing the host cell of the present invention in media under suitableconditions.
V] In another aspect, the present ion provides a use of the pharmaceutical composition ofthe present invention in the manufacture of a medicament for the treatment of a coagulopathy in at in need thereof.[0046AW] In a further aspect, the present invention provides a use of the recombinant factor VIII fusionprotein of the t ion in the manufacture of a medicament for the treatment of a coagulopathy.
INCORPORATION BY REFERENCE All publications, s, and patent applications mentioned in this specification are hereinincorporated by nce to the same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the invention may be further explained by reference to thefollowing detailed description and accompanying drawings that sets forth illustrative embodiments. shows a schematic representation of the FVIII architecture and spatial arrangement ofthe s during processing and ng, and is intended to represent both native FVIII and B domaindeleted variants. The Al domain ranges from residue 1 to 372 (numbering relative to the mature form ofFVIII sequence NCBI Protein RefSeq NP_000123 and encompassing a1 residues), A2 domain rangesfrom residue 373 to 740, B domain ranges from residue 741 to 1648, A3 domain ranges from residue1649 to 2019 (encompassing a3 acidic region), C1 domain ranges from 2020 to 2172, and the C2domain ranges from residue 2173 to 2332. BDD variants include deletions between the range 741 to'1648, leaving some or no remnant residues, with a non- limiting BDD remnant sequence beingSFSQNPPVLKRHQR (SEQ ID NO: 1614). shows the domain ecture of a single chainFVIII prior to processing. Arrows indicate the sites at residues R3 72, R740, R1648, and R1689 that arecleaved in the processing and conversion of FVIII to FVIIIa. shows the FVIII molecule that hasbeen processed into the heterodimer by the cleavage at the R1648 e, with the a3 acidic region of[Text continues on page 21]the A3 domain indicated on the N—terminus of the A3. shows the FVIII molecule processed intothe FVIIIa heterotrimer by the cleavage at the R372, R740, and R1689 residues. is a schematic of the coagulation e, showing the intrinsic and extrinsic armsleading to the common pathway. depicts the amino acid sequence of mature human factor VIII (SEQ ID NO: 1592). depicts a factor VIII sequence with a deletion of a portion of the B domain (SEQ IDNO: 1593). illustrates several examples of CFXTEN configurations of FVIII linked to XTEN (thelatter shown as thick, wavy lines). In all cases, the FVIII can be either native or a BDD form of FVIII, ora single chain form in which the entire B domain, including the native cleavage sites are removed. shows, left to right, three variations of single chain factor VIII with XTEN linked to the N—terminus,the C-terminus, and two XTEN linked to the N— and C-terminus. shows six variations of maturedimer FVIII with, left to right, an XTEN linked to the inus of the A1 domain; an XTENlinked to the C-terminus of the C2 domain; an XTEN linked to the N—terminus of the A1 domain and theC-terminus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain and to the N-us of the A3 domain; an XTEN linked to the C-terminus of the C2 domain and to the N-terminusof the A3 domain via residual B domain amino acids; and an XTEN linked to the N-terminus of the A1domain, the C-terminus of the A2 domain via residual B domain amino acids, and to the C-terminus ofthe C2 domain. shows, left to right, three variations of single chain factor VIII: an XTEN linkedto the N—terminus of the A1 , an XTEN linked within a surface loop of the A1 domain and anXTEN linked within a surface loop of the A3 domain; an XTEN linked within a surface loop of the A2domain, an XTEN linked within a surface loop of the C2 domain and an XTEN linked to the C terminusof the C2 domain; an XTEN linked to the N—terminus of the A1 domain and within a surface loop of theC1 domain and to the C-terminus of the C . shows six variations of mature heterodimerFVIII with, left to right, an XTEN linked to the N-terminus of the A1 domain, an XTEN linked within asurface loop of the A1 domain, and an XTEN linked within a surface loop of the A3 domain; an XTENlinked within a surface loop of the A2 domain, and an XTEN linked within a surface loop of the C1domain, and an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N—terminus ofthe A1 domain, an XTEN linked within a surface loop of the A1 domain, an XTEN linked within asurface loop of the A3 domain, and an XTEN linked to the C-terminus of the C2 ; an XTENlinked to the N—terminus of the A1 domain, an XTEN linked to the N—terminus of the A3 domain viaresidual amino acids of the B domain, and an XTEN linked within a surface loop of the C2 domain; anXTEN linked within a surface loop of the A2 , an XTEN linked to the N—terminus of the A3domain via al amino acids of the B domain, an XTEN linked within a surface loop of the C1domain, and an XTEN linked to the C-terminus of the C2 domain; and an XTEN linked within the Bdomain or between the residual B domain residues of the BDD variant (and the invention alsocontemplates a ion in which the XTEN replaces the entirety of the B , including all nativecleavage sites, linking the A2 and A3 domains, resulting in a single chain form of factor VIII). Thisfigure also embodies all variations in which one or more XTEN sequences are inserted within the Bdomain and the resulting fusions are cleaved at one or more sites (e.g., at R1648 site) during intracellularprocessing. is a graphic portrayal of a CFXTEN construct with an XTEN inserted within the Bdomain and linked to the C-terminus of the C2 domain rating the unstructured characteristic of theXTEN leading to random coil formation that can cover portions of the factor VIII proximal to the XTEN.
In the lower panel, the drawing s that when XTEN is in random coil, it can adopt a conformationresulting in steric hindrance that blocks binding of factor VIII inhibitor antibodies that would otherwisehave affinity for epitopes proximal to the XTEN site of insertion. is a graphic portrayal of the various analyses performed on a FVIII B-domain deletedce to identify insertion sites for XTEN within the FVIII sequence. Each of lines A—H are on anarbitrary scale of Y axis values across the FVIII BDD sequence such that low values represent areas witha high predicted tolerance for XTEN insertion, with the residue numbers on the X axis. Line A shows thedomain boundaries; all discontinuities in this line represent boundaries that are likely to accept XTEN.
Line B shows exon boundaries; i.e., each step in the line represents a new exon. Line C shown regionsthat were not visible in the X-ray structure due to a lack of order in the crystal. Lines labeled D representsle predictions of order that were calculated using the respective programs dex found on theWide web site bip.weizmann.ac.il/fidbin/findex (last accessed February 23, 2011) (see JaimePrilusky, Clifford E. Felder, Tzviya en-Mordehai, Edwin Rydberg, Oma Man, Jacques S.nn, Israel Silman, and Joel L. n, 2005, Bioinformatics based on the Kyte & llealgorithm, as well as RONN found on the Wide web site strubi.ox.ac.uk/RONN (last accessedry 23, 2011) (see Yang,Z.R., Thomson, R., McMeil, P. and Esnouf, R.M. (2005) RONN: the bio-basis function neural network technique applied to the ion of natively disordered regions inproteins Bioinformatics 21: 3369-3376. Lines E and F were calculated based on multiple cealignments of FVIII genes from 11 s available in GenBank. Line E represents the conservationof individual residues. Line F represent the conservation of 3 amino acid segments of FVIII. Lines G andH represent gaps and insertions observed in the le ce alignment of 11 mammalian FVIIIgenes. Line J lists the XTEN insertion points by amino acid number that were obtained based bycombining the multiple measurements above. depicts the sites in a FVIII B-domain deleted sequence (SEQ ID NO: 1594) identified asactive insertion points for XTEN using the information depicted in and as confirmed in the assaysof Example 34. depicts the range of sites in a FVIII B-domain deleted sequence (SEQ ID NO: 1595)identified for insertion ofXTEN using the information depicted in and or Example 34 plus a spanof amino acids around each insertion point that are considered suitable for insertion of XTEN. is a schematic of the assembly of a CFXTEN library created by identifying insertionpoints as described for FIGS. 7 followed by insertion of single XTEN (black bars) at the various insertionpoints using molecular biology techniques. The constructs are expressed and recovered, then evaluatedfor FVIII activity and pharmacokinetic properties to identify those CFXTEN configurations that result inenhanced ties. is a schematic of the assembly of a CFXTEN component library in which segments ofFVIII BDD domains, either singly or linked to various s ofXTEN (black bars) are assembled in acombinatorial fashion into libraries of genes encoding the CFXTEN, which can then be evaluated forFVIII actiVity and pharmacokinetic properties to identify those CFXTEN configurations that result inenhanced properties. illustrates several examples of CFXTEN configurations with XTEN (shown as thick,wavy lines), with certain XTEN releasable by inserting cleavage sequences (indicated by black triangles)that are cleavable by procoagulant ses. A illustrates a scFVIII with two terminal releasableXTENS. B illustrates the same configuration as A but with an onal non-releasableXTEN linking the A3 and C1 domains. C illustrates a mature heterodimer FVIII with twoterminal releasable XTEN. D illustrates the same configuration as 10C but with an additionalnon-releasable XTEN linking the A3 and C1 domains. is a tic rt of representative steps in the assembly, production and thetion of an XTEN. is a schematic flowchart of representative steps in the assembly of a CFXTENpolynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are annealed intosequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequencemotifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well asligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. Theresulting pool of ligation products is gel-purified and the band with the desired length ofXTEN is cut,resulting in an isolated XTEN gene with a stopper ce 505. The XTEN gene is cloned into a stuffervector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion isthen performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resultingproduct is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, ingin the gene 500 encoding an FVIII-XTEN fusion n. is a tic flowchart of representative steps in the assembly of a gene encodingfusion protein comprising a CF and XTEN, its expression and ry as a fusion protein, and itsevaluation as a candidate CFXTEN product. illustrates the use of donor XTEN sequences to produce ted XTENs. Aprovides the sequence of AG864 (SEQ ID NO: 1596), with the underlined sequence used to generate asequence length of 576 (SEQ ID NO: 1597). B provides the sequence of AG864 (SEQ ID NO:1598), with the ined sequence used to generate a ce length of 288 (SEQ ID NO: 1599).
C provides the sequence of AG864 (SEQ ID NO: 1600), with the underlined sequence used togenerate a sequence length of 144 (SEQ ID NO: 1601). D provides the sequence of AE864 (SEQID NO: 1602), with the underlined sequence used to generate a sequence length of 576 (SEQ ID NO:1603). E provides the sequence of AE864 (SEQ ID NO: 1604), with the underlined sequenceWO 22617 used to generate a sequence length of 288 (SEQ ID NO: 1605). F provides the sequence ofAE864 (SEQ ID NO: 1606) used to generate four sequences of 144 length (SEQ ID NOS 1607-1610,respectively, in order of ance) (the double underline indicates the first amino acid in the 144sequence with the single underline representing the balance of that sequence). is a schematic representation of the design of Factor VIII-XTEN expression vectorswith different strategies introducing XTEN elements into the FVIII coding sequence. A shows anexpression vector ng XTEN fused to the 3’ end of the sequence encoding FVIII. B depictsan sion vector encoding an XTEN element inserted into the middle of the coding sequenceencoding a single FVIII. C depicts an expression vector encoding two XTEN elements: oneinserted internal to the FVIII coding sequence, and the other fused to the 3’ end of the FVIII codingsequence. rates the process of combinatorial gene assembly of genes encoding XTEN. Inthis case, the genes are assembled from 6 base fragments and each fragment is available in 4 differentcodon versions (A, B, C and D). This allows for a theoretical diversity of 4096 in the assembly of a 12amino acid motif. shows the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeysafter single doses of different compositions of GFP linked to unstructured polypeptides of varying length,administered either subcutaneously or intravenously, as described in Example 41. The compositionswere GFP-L288, 76, GFP-XTEN_AF576, GFP-Y576 and D836-GFP. Blood sampleswere analyzed at various times after injection and the tration of GFP in plasma was measured byELISA using a polyclonal antibody t GFP for capture and a biotinylated preparation of the samepolyclonal antibody for detection. Results are presented as the plasma concentration versus time (h) afterdosing and show, in particular, a considerable increase in half-life for the D836-GFP, thecomposition with the longest sequence length of XTEN. The construct with the shortest sequence length,the GFP-L288 had the shortest half-life. shows an SDS-PAGE gel of samples from a stability study of the fusion protein ofXTEN_AE864 fused to the inus of GFP (see Example 42). The GFP-XTEN was ted incynomolgus plasma and rat kidney lysate for up to 7 days at 37°C. In addition, GFP-XTEN administeredto cynomolgus monkeys was also assessed. Samples were withdrawn at O, 1 and 7 days and analyzed bySDS PAGE ed by detection using Western analysis with dies against GFP. shows results of a size exclusion chromatography analysis of glucagon-XTENconstruct samples measured against protein standards of known molecular weight, with the graph outputas absorbance versus retention volume, as described in Example 40. The on-XTEN constructs are1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and 4) on-Y36. The s indicate anincrease in apparent molecular weight with increasing length ofXTEN moiety (see Example 40 for data). shows results of a Western blot of proteins expressed by cell culture of cellstransformed with constructs as designated (Example 25). The samples in lanes 1-12 were: MWStandards, FVIII (42.5 ng), pBCOIOOB, pBC0114A, pBC0100, pBC0114, pBC0135, pBC0136,pBC0137, pBC0145, 9, and pBC0146, respectively. Lanes 8, 9 and 12 show bands consistentwith a FVIII with a C-terminal XTEN288, with an estimated MW of 95 kDa. Lanes 7 and 11 showbands consistent with a FVIII with a C-terminal XTEN42, with an estimated MW of 175 kDa. Lanes 2-6show bands consistent with FVIII and heavy chain. Lanes 10 and 23 show bands consistent with heavychain. Lane 7 shows a band consistent with heavy chain and an attached XTEN42. shows the results of FVIII assay on samples obtained from FVIII and von Willebrandfactor double knock-out mice with hydrodynamic plasmid DNA injection, as detailed in e 36. is a graphic and tabular portrayal of the pharmacokinetic properties of rBDD-FVIII andthe purified CFXTEN fusion proteins pBC0145 and pBC0146 (with C-terminal XTEN) stered toeither HemA or FVIII/VWF double knock-out mice as bed in Example 30, showing the enhancedhalf-life of the CFXTEN in both strains of mice. is a graphic and tabular portrayal of the pharmacokinetic properties of rBDD-FVIII andthe CFXTEN fusion proteins pSD0050 and pSDOO62 (with internal inserted XTEN) administered toeither HemA (A) or FVIII/VWF double knock-out mice (B) using a cell culture PK assayin HemA mice. Dose, 5-minute recovery, and half-life (Tl/2) are shown, as described in Example 32,underscoring the enhanced recovery and half-life of the CFXTEN ed to the positive l FVIIIin both strains of mice. is a graphic depiction of a titration of GMA8021 FVIII inhibitor using the pBC0114BDD-FVIII AND CFXTEN construct 9.002 with three 144 amino acid XTEN insertions ates 18, 745 and 2332. The data indicate a right-shift of imately 0.7 order of magnitude inthe amount of antibody in [Lg/ml required to inhibit the CFXTEN to the 50% level, compared to FVIIIpositive control. is a schematic of the logic flow chart of the algorithm SegScore. In the figure thefollowing legend applies: i, j - counters used in the control loops that run through the entire sequence;HitCount- this variable is a counter that keeps track of how many times a subsequence encounters anidentical subsequence in a block; SubSeqX - this variable holds the subsequence that is being checked forredundancy; SubSeqY - this variable holds the subsequence that the SubSeqX is checked against;en - this variable holds the user determined length of the block; SegLen - this variable holds thelength of a t. The program is hardcoded to generate scores for subsequences of s 3, 4, 5, 6,7, 8, 9, and 10; Block - this le holds a string of length en. The string is composed of lettersfrom an input XTEN sequence and is ined by the position of the i counter; SubSeqList - this is alist that holds all of the generated subsequence scores. depicts the application of the algorithm SegScore to a hypothetical XTEN of 11 aminoacids (SEQ ID NO: 1591) in order to determine the repetitiveness. An XTEN sequence consisting ofNamino acids is divided into N—S+1 subsequences of length S (S=3 in this case). A pair-wise comparisonof all subsequences is performed and the average number of identical subsequences is calculated to resultin the subsequence score of 1.89. is a graph of the individual construct values of the ratio of FVIII activity in the assayedCFXTEN to that of the pBC114 FVIII ve control after exposure to the GMA8021 antibody toFVIII, grouped ing to the number ofXTEN in the construct fusion protein (see Example 28). Thes show an essentially linear relationship in the ability of the CFXTEN to retain FVIII actiVity withincreasing number of incorporated XTEN. depicts the primary sequence and domain structure of mature B-domain deleted (BDD)human FVIII construct (Example 46). The location of the introduced NheI and Clal restriction sites isshown. Note that the amino acid numbering corresponds to the amino acid positions in the primarysequence of mature FVIII (). Individual domains are bounded by gray lines/boxes with domainidentification in gray text. Acidic regions (a1, a2, a3) are indicated with dashed boxes. Solidwedges/triangles indicate sites of thrombin ge in the activation of FVIII to . Unfilled/triangle indicates the site of intracellular proteolytic processing to the ained form of FVIII.
Hexagons indicate sites of N—linked glycosylation. Circles indicate sites of Tyr sulfation. Unique non-native restriction sites (Nhel, GCTAG; Clal, ATCGAT) introduced into cDNA to facilitate XTENinsertion/recombination are highlighted in gray with double ine. provides graphical representation of the FVIII construct described in ,indicating the domain organization and the location of native and non-native restriction sites. shows the graphical ASAView outputs for structural datasets 2R7E, 3CDZ, andPMOO76106. Accessible Solvent Areas (ASA) for the amino acids in domains A1, A2, A3, C1 and C2are shown. Analyses were performed on X-ray crystallographic coordinates 3CDZ (Ngo et al., Structure16: 597-606 (2008)) and 2R7E (Shen et al., Blood 111:1240-1247 ) deposited in the Protein DataBank maintained by the Research oratory for Structural Bioinformatics (RCSB;/www.rcsb.org/pdb), as well as on atomic coordinates PMOO76106 for the predicted refined FVIIIstructure derived from a molecular dynamics simulation study (Venkateswarlu, BMC Struct. Biol. 10:7(2010)) deposited in the n Model Database (http://mi.caspur.it/PMDB/main.php) maintained byConsorzio Interuniversitario per le Applicazioni di Supercalcolo per Universita e Riserca (CASPUR) andthe Department of Biochemical Sciences of the University of Rome. shows a structural representation of the on ofXTEN insertion sites. The centraldrawing corresponding to the crystal structure of FVIII (PDB: 2R7E) is surrounded by detailed View ofdomains A1, A2, A3, C1 and C2. Beta strands and alpha helices are shown as ribbon representation.
Loops are shown as alpha carbon pipes. The amino acids at XTEN ion sites are shown as CPKsphere representation. The number in each graph indicate the location of the XTEN insertion sitesaccording to the numbering in . shows a structural entation of the location ofXTEN insertion sites shown in wherein the resulting recombinant FVIII protein displays FVIII ty. shows a structural representation of the location ofXTEN insertion sites shown in wherein the resulting recombinant FVIII protein displays FVIII actiVity. shows a structural representation of the location ofXTEN insertion sites shown in wherein the resulting recombinant FVIII n displays FVIII activity. shows a ClustalW multiple sequence ent of domains A], A2, A3, C1 and C2 ofFVIII showing the on ofXTEN insertions resulting in recombinant FVIII proteins displaying FVIIIactiVity (black box, white text) or displaying no FVIII actiVity (grey box, bold text). shows a DSSP graphical representation of the ary structure of the twopolypeptide chains in a native active human FVIII crystal ure deposited under the identifier 2R7E atthe Protein Data Bank (see Example 47). Amino acid sequence numbering is the same as in the proteinsequence in . The beta sheet regions are shown as filled arrows and are designated B1 to B66.
The location of the XTEN permissive loops is denoted by crosshatched boxes. Domain Al XTENpermissive loops are designated Loop Al-l and Loop Al -2. Domain A2 XTEN permissive loops aredesignated Loop A2-l and Loop A2-2. Domain A3 XTEN permissive loops are designated Loop A3-land Loop A3-2. shows a DSSP graphical representation of the secondary ure of the twopolypeptide chains in a native active human FVIII crystal structure deposited under the identifier 2R7E atthe Protein Data Bank (see Example 47). Amino acid sequence numbering is the same as in the proteinsequence in . The beta sheet regions are shown as filled arrows and are designated B1 to B66.
The location of the XTEN permissive loops is denoted by crosshatched boxes. Domain Al XTENpermissive loops are designated Loop Al-l and Loop Al -2. Domain A2 XTEN permissive loops aredesignated Loop A2-l and Loop A2-2. Domain A3 XTEN permissive loops are designated Loop A3-land Loop A3-2. shows a ClustalW multiple sequence alignment of domains A], A2, A3, C1 and C2 ofFVIII showing the location ofXTEN insertions resulting in recombinant FVIII proteins displaying FVIIIactiVity (black box, white text) or displaying no FVIII actiVity (grey box, bold text). The locations of theXTEN permissive loops are indicated by dashed rectangles (see Example 47).
. A presents a front View ural representation of human FVIII R7E)showing the location of domains Al, A2, A3, C1 and C2 (circled in dashed lined) and the locations ofXTEN permissive loops Al -1, Al -2, A2-l, A2-2, A3-l and A3-2 highlighted as CPK sphererepresentations. B presents a side View structural representation of human FVIII (PDB:2R7E)g the location of domains Al, A2, A3, C1 and C2 (circled in dashed lined) and the locations ofXTEN sive loops Al -1, Al -2, A2-l, A2-2, A3-l and A3-2 highlighted as CPK sphereentations. shows the top View structural representations of ed human FVIII (PDB:2R7E) Adomains showing the location ofXTEN sive loops highlighted as CPK sphere representations.
B, 42D and 42F show side View structural representations of isolated human FVIII (PDB:2R7E)A domains showing the location ofXTEN permissive loops highlighted as CPK sphere representations. shows sequences of various factor VIII in deletions and individual mutations.
Lines 4-10 show various B-domain deletions with indicated XTEN linking the flanking B-domainresidual or A3 domain residues. The R1648A on is indicated by arrow in line 5 and 8, while theY] 68OF mutation is ted by arrow in lines 8-10. is a bar graph of chromogenic and aPTT assay activity of various CFXTEN with singleXTEN insertions (Example 49). is a bar graph of chromogenic and aPTT assay actiVity of various CFXTEN with 2XTEN insertions (Example 49).
FIG 46 is a bar graph of chromogenic and aPTT assay actiVity of various CFXTEN with 3XTEN insertions (Example 49). is a graph of plasma levels in DKO mice of various stered CFXTEN with singleXTEN insertions compared to a BDD-FVIII l, demonstrating the 10- to d longer half-lifeachieved by the XTEN insertions at various locations (Example 50). is a graph of plasma levels in DKO mice of various administered CFXTEN with one,two, and three XTEN insertions ed to a BDD-FVIII control, demonstrating the increases in half-life achieved by the inclusion of additional XTEN insertions compared to single or two insertions(Example 51). are graphs of the plotted inhbition curves for remaining factor VIII procoagulantactiVity in samples assayed in the Bethesda assay with three hemophilia patient sera (FIGS. 49A—C) orsheep anti-FVII (D) described in Example 52, demonstrating a clear left-shift of the inhibitioncurve for the two CFXTEN molecules compared to the FVIII not linked to XTEN.
DETAILED PTION OF THE INVENTION Before the ments of the ion are described, it is to be understood that suchembodiments are ed by way of example only, and that various alternatives to the embodiments ofthe invention described herein may be employed in practicing the ion. Numerous variations,changes, and substitutions will now occur to those skilled in the art without departing from the invention.
Unless ise defined, all technical and scientific terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials are described below. In case of conflict,the patent specification, including definitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Numerous variations, changes, andsubstitutions will now occur to those skilled in the art t departing from the ion.
DEFINITIONS] In the context of the present application, the following terms have the meanings ascribed tothem unless specified otherwise:] As used in the specification and claims, the singular forms 66 39 66a an” and “the” include pluralreferences unless the context clearly dictates ise. For example, the term “a cell” includes aplurality of cells, including mixtures thereof.
The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer topolymers of amino acids of any length. The polymer may be linear or branched, it may comprisemodified amino acids, and it may be interrupted by non-amino acids. The terms also encompass anamino acid polymer that has been d, for example, by disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation With a labelingcomponent.
As used herein, the term “amino acid” refers to either natural and/or ral or syntheticamino acids, including but not limited to both the D or L optical isomers, and amino acid analogs andpeptidomimetics. Standard single or three letter codes are used to designate amino acids.
The term "domain," When used in reference to a factor VIII polypeptide refers to either a fulllength domain or a functional fragment thereof, for example, full length or onal fragments of theA1 domain, A2 domain, A3 domain, B domain, C1 domain, and/or C2 domain of factor VIII.
] The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P),alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F),tyrosine (Y), tryptophan (W), ine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N),glutamic acid (E), ic acid (D), serine (S), and threonine (T).
The term “non-naturally occurring,” as applied to sequences and as used herein, meanspolypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, ordo not have a high degree of homology With a Wild-type or naturally-occurring sequence found in amammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%,98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to anatural sequence When suitably d.
The terms “hydrophilic” and “hydrophobic” refer to the degree of affinity that a substance hasWith water. A hydrophilic substance has a strong y for water, tending to dissolve in, mix With, orbe wetted by water, While a hydrophobic substance substantially lacks affinity for water, tending to repeland not absorb water and tending not to dissolve in or mix With or be wetted by water. Amino acids canbe characterized based on their hydrophobicity. A number of scales have been developed. An exampleis a scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is listed in Hopp, TP, et al.,Proc Natl Acad Sci U S A (1981) 78:3 824. Examples of “hydrophilic amino acids” are ne, ,threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acidsaspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan,tyrosine, phenylalanine, methionine, e, cine, and valine.
A ent” When applied to a protein, is a truncated form of a native biologically activen that retains at least a portion of the therapeutic and/or biological activity. A “variant”. Whenapplied to a protein is a protein With ce homology to the native biologically active protein that 2012/046326retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. Forexample, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%amino acid sequence identity compared with the reference ically active protein. As used herein,the term “biologically active protein moiety” includes proteins modified deliberately, as for example, bysite directed nesis, synthesis of the encoding gene, insertions, or accidentally through mutations.
The term “sequence variant” means polypeptides that have been modified compared to theirnative or original sequence by one or more amino acid insertions, deletions, or substitutions. Insertionsmay be located at either or botlt termini of the n, and/or may be positioned within internal regionsof the amino acid sequence. A non—limiting example is insertion of an XTEN sequence within thesequence ot‘the biologically—active payload protein. In deletion variants, one or more amino acidresidues in a polypeptide as described herein are removed. Deletion variants, therefore, include allfragments of a payload polypeptide sequence. in substitution ts, one or more amino acid residuesof a polypeptide are d and replaced with alternative residues. in one aspect, the substitutions areconservative in nature and conservative substitutions of this type are well known in the art.
] As used herein, “internal XTEN” refers to XTEN sequences that have been inserted into thesequence of the coagulation factor. Internal XTENs can be constructed by insertion of an XTENce into the sequence of a ation factor such as FVIII, either by insertion between twont amino acids within a domain (“intradomain”) or between two domains (“interdomain”) of thecoagulation factor or wherein XTEN replaces a partial, internal sequence of the coagulation factor.
As used herein, “terminal XTE ” refers to XTENsequences that have been fused to or in theN— or C-terminus of the coagulation factor or to a proteolytic cleavage sequence or linker at the N— or C-terminus of the coagulation factor. Terminal XTENs can be fused to the native termini of thecoagulation factor. Alternatively, terminal XTENs can replace a portion of a terminal sequence of thecoagulation factor.
] The term “XTEN release site” refers to a cleavage ce in CFXTEN fusion proteins thatcan be recognized and cleaved by a mammalian protease, effecting release of an XTEN or a portion of anXTEN from the CFXTEN fusion protein. As used herein, lian protease” means a protease thatnormally exists in the body fluids, cells or tissues of a mammal. XTEN release sites can be engineered tobe cleaved by various mammalian proteases (a.k.a. “XTEN e proteases”) such as FXIa, FXIIa,rein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-l7, MMP-20, orany protease that is present during a clotting event. Other equivalent proteases enous orexogenous) that are capable of recognizing a defined cleavage site can be utilized. The cleavage sitescan be adjusted and tailored to the protease utilized.
] The term “within”, when referring to a first polypeptide being linked to a second polypeptide,encompasses linking that connects the N—terminus of the first or second polypeptide to the inus ofthe second or first polypeptide, respectively, as well as insertion of the first polypeptide into the ceof the second polypeptide. For example, when an XTEN is linked “within” a domain of a factor VIIIpolypeptide, the XTEN may be linked to the N—terminus, the C-terminus, or may be inserted in saiddomain.
As used , the term “site,” when used to refer to an insertion site of an XTEN within or toa biological polypeptide such as a factor VIII, represents the amino acid position at which the XTEN islinked. When numbered sites are described, such as a first, second, third, fourth, fifth, or sixth site for theinsertion of an XTEN within or to the factor VIII, each site will be understood to ent a ct sitein the factor VIII; e.g., the second site is a different factor VIII location from the first site, the third site isent from the second and the first, etc.
“Activity” or agulant activity” as applied to ) of a CFXTEN polypeptide providedherein, refers to the ability to bind to a target coagulation protein substrate or cofactor and promote aclotting event, r measured by an in vitro, ex vivo or in vivo assay. Such assays include, but are notlimited to, one-stage clotting assays, two-stage clotting assays, chromogenic assays, and ELISA assays.
“Biological actiVity” refers to an in vitro or in vivo biological function or effect, including but not limitedto either or or ligand binding, or an effect on ation lly known in the art for the FVIIIcoagulation factor, or a cellular, physiologic, or clinical response, including arrest of a bleeding episode.
As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay asdescribed herein or as otherwise known in the art.
A “host cell” includes an individual cell or cell culture which can be or has been a recipient forthe subject vectors. Host cells include progeny of a single host cell. The progeny may not necessarily becompletely identical (in morphology or in genomic of total DNA complement) to the al parent celldue to natural, accidental, or deliberate on. A host cell includes cells transfected in Vivo with avector of this invention.
“Isolated” when used to describe the various polypeptides disclosed herein, means ptidethat has been identified and separated and/or recovered from a component of its natural environment.
Contaminant components of its natural environment are materials that would typically interfere withdiagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturallyoccurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not e“isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”,“separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments f, isdistinguishable from its naturally occurring counterpart in that the concentration or number of moleculesper volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptidemade by recombinant means and expressed in a host cell is considered to be “isolated.”] An “isolated” polynucleotide or polypeptide-encoding nucleic acid or other polypeptide-encoding c acid is a nucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associated in the natural source of thepolypeptide-encoding c acid. An isolated polypeptide-encoding nucleic acid le is other thanin the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acidmolecules therefore are distinguished from the c polypeptide-encoding nucleic acid molecule as itexists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptidewhere, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal ondifferent from that of natural cells.
A ric” protein contains at least one fusion polypeptide comprising at least one region ina different position in the sequence than that which occurs in . The regions may normally exist inseparate proteins and are brought together in the fusion polypeptide; or they may normally exist in thesame protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may bed, for example, by chemical synthesis, or by creating and translating a polynucleotide in which thepeptide regions are encoded in the desired relationship.
“Conjugated”, “linked,” “fused,” and “fusion” are used interchangeably herein. These termsrefer to the joining together of two or more chemical elements, sequences or components, by whatevermeans ing chemical ation or recombinant means. For example, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription of the sequence. Generally, blylinked” means that the DNA sequences being linked are contiguous, and in reading phase or in-frame.
An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form acontinuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
Thus, the ing recombinant fusion protein is a single protein containing two or more segments thatpond to polypeptides encoded by the original ORFs (which segments are not normally so joined innature).
] In the context of polypeptides, a “linear sequence” or a nce” is an order of amino acidsin a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other inthe sequence are contiguous in the y structure of the polypeptide. A “partial sequence” is a linearsequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
“Heterologous” means derived from a genotypically distinct entity from the rest of the entity towhich it is being compared. For example, a glycine rich sequence d from its native codingsequence and operatively linked to a coding sequence other than the native sequence is a heterologouse rich sequence. The term “heterologous” as applied to a cleotide, a polypeptide, means thatthe polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest ofthe entity to which it is being compared.
The terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are usedinterchangeably. They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting es of polynucleotides: coding or ding regions of a gene or gene fragment, loci(locus) defined from linkage analysis, exons, introns, messenger RNA , transfer RNA, ribosomalRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, 2012/046326isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Apolynucleotide may comprise modified tides, such as methylated nucleotides and tideanalogs. If t, modifications to the nucleotide structure may be imparted before or after assembly ofthe polymer. The ce of nucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as by conjugation with a labelingcomponent.
The term “complement of a polynucleotide” denotes a polynucleotide molecule having amentary base sequence and reverse ation as compared to a reference sequence, such that itcould hybridize with a reference sequence with complete y.
“Recombinant” as applied to a polynucleotide means that the polynucleotide is the product ofvarious combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that resultin a construct that can potentially be expressed as a inant protein in a host cell.
The terms “gene” and “gene fragment” are used interchangeably herein. They refer to apolynucleotide ning at least one open reading frame that is capable of encoding a particular proteinafter being ribed and translated. A gene or gene fragment may be genomic or cDNA, as long as thepolynucleotide contains at least one open g frame, which may cover the entire coding region or asegment thereof A “fusion gene” is a gene composed of at least two heterologous polynucleotides thatare linked together.
“Homology” or “homologous” or “sequence identity” refers to sequence similarity orinterchangeability between two or more cleotide sequences or between two or more polypeptidesequences. When using a program such as BestFit to determine sequence identity, rity orhomology between two different amino acid sequences, the default settings may be used, or anappropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity,similarity or homology scores. Preferably, polynucleotides that are homologous are those whichhybridize under ent conditions as defined herein and have at least 70%, preferably at least 80%,more ably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, andeven more ably 99% sequence identity compared to those sequences. Polypeptides that arehomologous preferably have sequence identities that are at least 70%, ably at least 80%, even morepreferably at least 90%, even more preferably at least 95-99%, and most preferably 100% identical.
”Ligation” refers to the process of forming phosphodiester bonds between two nucleic acidfragments or genes, linking them together. To ligate the DNA fragments or genes together, the ends ofthe DNA must be compatible with each other. In some cases, the ends will be directly compatible afterendonuclease digestion. However, it may be necessary to first convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make them compatible for ligation.
The terms “stringent conditions” or gent hybridization conditions” includes reference toconditions under which a polynucleotide will hybridize to its target sequence, to a detectably rdegree than other sequences (e.g., at least 2-fold over background). Generally, stringency ofhybridization is expressed, in part, with reference to the temperature and salt concentration under whichthe wash step is carried out. lly, stringent conditions will be those in which the salt trationis less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH7.0 to 8.3 and the temperature is at least about 30°C for short polynucleotides (e. g., 10 to 50 nucleotides)and at least about 60°C for long polynucleotides (e. g., greater than 50 nucleotides)—for example,“stringent conditions” can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, andthree washes for 15 min each in SC/1% SDS at 60°C to 65°C. atively, temperatures of about65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2><SSC,with SDS being present at about 0.1%. Such wash temperatures are typically selected to be about 5°C to°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acidhybridization are well known and can be found in Sambrook, J. et a]. “Molecular Cloning: A LaboratoryManual,” 3ml edition, Cold Spring Harbor Laboratory Press, 2001. Typically, blocking reagents are usedto block non-specific hybridization. Such blocking reagents include, for instance, sheared and denaturedsalmon sperm DNA at about 0 ug/ml. Organic solvent, such as formamide at a concentration ofabout 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNAhybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinaryskill in the art.
The terms “percent identity,” tage of sequence identity,” and “% identity,” as applied topolynucleotide ces, refer to the percentage of residue matches between at least two polynucleotidesequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized andreproducible way, gaps in the sequences being compared in order to optimize alignment between twosequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identitymay be measured over the length of an entire defined cleotide sequence, or may be measured overa shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotidesequence, for ce, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least210 or at least 450 contiguous es. Such lengths are exemplary only, and it is understood that anyfragment length supported by the ces shown herein, in the tables, figures or Sequence Listing, maybe used to describe a length over which percentage identity may be ed. The percentage ofsequence identity is calculated by comparing two optimally aligned sequences over the window ofcomparison, determining the number of matched ons (at which identical residues occur in bothpolypeptide sequences), dividing the number of matched positions by the total number of positions in thewindow of comparison (i.e., the window size), and lying the result by 100 to yield the percentageof sequence identity. When sequences of different length are to be compared, the st sequencedefines the length of the window of comparison. Conservative substitutions are not ered whencalculating ce identity.
“Percent (%) sequence identity,” with t to the polypeptide sequences identified herein, isdefined as the percentage of amino acid residues in a query sequence that are identical with the aminoacid es of a second, nce polypeptide sequence or a portion thereof, after aligning theces and introducing gaps, if necessary, to achieve the maximum percent ce identity, and notconsidering any conservative substitutions as part of the sequence identity. Alignment for purposes ofdetermining percent amino acid sequence ty can be achieved in various ways that are within theskill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2,ALIGN or Megalign (DNASTAR) re. Those skilled in the art can ine appropriateparameters for measuring alignment, including any thms needed to achieve maximal alignmentover the full length of the sequences being compared. Percent ty may be measured over the lengthof an entire defined polypeptide sequence, or may be measured over a r length, for example, overthe length of a nt taken from a larger, defined polypeptide sequence, for instance, a fragment of atleast 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
Such lengths are exemplary only, and it is understood that any fragment length supported by thesequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length overwhich percentage identity may be ed.
The term “non-repetitiveness” as used herein in the t of a polypeptide refers to a lack orlimited degree of internal homology in a peptide or polypeptide sequence. The term “substantially non-repetitive” can mean, for example, that there are few or no instances of four contiguous amino acids inthe sequence that are identical amino acid types or that the polypeptide has a subsequence score (definedinfra) of 10 or less or that there is no a pattern in the order, from N- to C-terminus, of the sequence motifsthat constitute the polypeptide sequence. The term “repetitiveness” as used herein in the context of apolypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, aitive” sequence may contain le identical copies of short amino acid sequences. For instance,a polypeptide sequence of interest may be divided into n-mer sequences and the number of identicalsequences can be counted. Highly tive sequences contain a large fraction of identical sequenceswhile non-repetitive sequences contain few identical sequences. In the context of a ptide, asequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, inwhich the motifs themselves have non-repetitive sequences, rendering the full-length polypeptidesubstantially non-repetitive. The length of polypeptide within which the non-repetitiveness is measuredcan vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or fromabout 9 to about 14 amino acids. “Repetitiveness” used in the context of polynucleotide sequences refersto the degree of internal homology in the sequence such as, for example, the frequency of identicalnucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing thefrequency of identical sequences.
A “vector” is a c acid le, preferably self-replicating in an appropriate host, whichtransfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors thatfunction ily for insertion of DNA or RNA into a cell, replication of vectors that function primarilyfor the ation of DNA or RNA, and expression vectors that function for transcription and/ortranslation of the DNA or RNA. Also included are vectors that provide more than one of the abovefunctions. An “expression vector” is a polynucleotide which, when introduced into an appropriate hostcell, can be ribed and translated into a polypeptide(s). An “expression system” y connotes ale host cell comprised of an expression vector that can function to yield a desired expressionproduct.
“Serum degradation resistance,” as applied to a polypeptide, refers to the ability of thepolypeptides to withstand degradation in blood or components thereof, which typically involvesproteases in the serum or plasma. The serum ation resistance can be measured by combining theprotein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range ofdays (e.g. 0.25, 0.5, l, 2, 4, 8, 16 days), typically at about 37°C. The samples for these time points can berun on a Western blot assay and the protein is detected with an antibody. The antibody can be to a tag inthe protein. If the protein shows a single band on the western, where the protein’s size is identical to thatof the injected protein, then no ation has occurred. In this exemplary method, the time pointwhere 50% of the protein is degraded, as judged by Western blots or equivalent techniques, is the serumdegradation half-life or “serum half-life” of the protein.
The term “t1/2 ” as used herein means the terminal half-life calculated as ln(2)/Kel. K61 is theterminal elimination rate constant calculated by linear regression of the terminal linear portion of the logconcentration vs. time curve. ife typically refers to the time required for half the quantity of anadministered substance ted in a living organism to be metabolized or eliminated by normalbiological processes. The terms “t1/239 ccterminal half-life39 cc elimination half-life” and “circulating half-, ,life” are used interchangeably herein.
“Active clearance” means the mechanisms by which a protein is removed from the circulationother than by filtration or coagulation, and which includes removal from the circulation mediated bycells, receptors, lism, or ation of the protein.
“Apparent molecular weight factor” and ent molecular weight” are related termsreferring to a measure of the relative increase or se in apparent lar weight exhibited by aparticular amino acid sequence. The apparent molecular weight is ined using size exclusionchromatography (SEC) or similar methods by comparing to globular protein standards, and is measuredin “apparent kD” units. The apparent molecular weight factor is the ratio between the apparent molecularweight and the actual molecular weight; the latter predicted by adding, based on amino acid composition,the ated molecular weight of each type of amino acid in the composition or by estimation fromison to molecular weight standards in an SDS electrophoresis gel.
The terms “hydrodynamic radius” or “Stokes ” is the effective radius (R}1 in nm) of amolecule in a solution measured by assuming that it is a body moving through the solution and resistedby the solution’s viscosity. In the embodiments of the invention, the hydrodynamic radius measurementsof the XTEN fusion ns correlate with the ‘apparent lar weight factor’, which is a moreintuitive measure. The “hydrodynamic radius” of a n affects its rate of diffusion in aqueoussolution as well as its ability to migrate in gels of olecules. The hydrodynamic radius of aprotein is determined by its molecular weight as well as by its structure, including shape andtness. s for determining the hydrodynamic radius are well known in the art, such as bythe use of size exclusion chromatography (SEC), as described in US. Patent Nos. 6,406,632 and7,294,513. Most proteins have ar ure, which is the most compact three-dimensional structurea protein can have with the smallest hydrodynamic radius. Some proteins adopt a random and open,unstructured, or ‘linear’ conformation and as a result have a much larger hydrodynamic radius comparedto typical globular proteins of similar molecular weight.
“Physiological conditions” refers to a set of conditions in a living host as well as in vitroconditions, including temperature, salt tration, pH, that mimic those conditions of a living subject.
A host of physiologically nt conditions for use in in vitro assays have been established. Generally,a physiological buffer contains a logical concentration of salt and is adjusted to a neutral pHranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety ofphysiological s are listed in Sambrook et al. (2001). Physiologically relevant temperature rangesfrom about 250C to about 380C, and preferably from about 350C to about 370C.
A “reactive group” is a chemical structure that can be coupled to a second reactive group.
Examples for reactive groups are amino groups, carboxyl groups, sulfhydryl , hydroxyl groups,aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with asecond reactive group. miting examples for activation are the reaction of a carboxyl group withcarbodiimide, the conversion of a carboxyl group into an activated ester, or the conversion of a carboxylgroup into an azide function.
“Controlled release agent”, “slow release agent”, “depot formulation” and “sustained releaseagent” are used interchangeably to refer to an agent capable of extending the duration of release of apolypeptide of the invention ve to the duration of release when the polypeptide is administered inthe absence of agent. Different embodiments of the present invention may have different release rates,resulting in different therapeutic amounts.
The terms “antigen”, t antigen” and “immunogen” are used interchangeably herein torefer to the structure or g determinant that an antibody fragment or an antibody fragment-basedeutic binds to or has specificity against.
] The term “payload” as used herein refers to a protein or peptide sequence that has biological oreutic activity; the counterpart to the pharmacophore of small molecules. Examples of payloadse, but are not limited to, coagulation factors, cytokines, enzymes, hormones, and blood and grthfactors.
The term “antagonist”, as used herein, includes any molecule that partially or fully blocks,inhibits, or neutralizes a biological activity of a native ptide disclosed herein. Methods foridentifying antagonists of a polypeptide may comprise ting a native polypeptide with a candidateantagonist molecule and measuring a able change in one or more biological activities normallyassociated with the native polypeptide. In the context of the present invention, antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any other molecules that decrease the effect of abiologically active protein.
] The term “agonist” is used in the broadest sense and includes any molecule that mimics abiological activity of a native polypeptide disclosed . Suitable t molecules specificallyinclude agonist antibodies or antibody fragments, nts or amino acid sequence variants of nativepolypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a nativepolypeptide may se contacting a native polypeptide with a candidate agonist le andmeasuring a detectable change in one or more biological activities normally associated with the nativepolypeptide.
As used herein, “treat” or “treating,” or “palliating” or “ameliorating” are used interchangeablyand mean stering a drug or a biologic to achieve a therapeutic benefit, to cure or reduce theseverity of an ng condition, or to achieve a prophylactic benefit, prevent or reduce the likelihood ofonset or severity the occurrence of a condition. By therapeutic benefit is meant eradication oramelioration of the underlying condition being treated or one or more of the logical symptomsassociated with the ying condition such that an improvement is observed in the subject,notwithstanding that the subject may still be afflicted with the underlying condition.
A “therapeutic effect” or “therapeutic benefit,” as used herein, refers to a logic effect,including but not limited to the mitigation, amelioration, or prevention of disease in humans or others, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting fromadministration of a fusion protein of the ion other than the ability to induce the production of anantibody against an antigenic epitope possessed by the biologically active protein. For prophylactic, the compositions may be administered to a subject at risk of developing a ular e,condition or symptom of the disease (e. g., a bleed in a diagnosed hemophilia A subject), or to a subjectreporting one or more of the physiological symptoms of a disease, even though a diagnosis of this diseasemay not have been made.
The terms “therapeutically effective amount” and “therapeutically effective dose”, as usedherein, refer to an amount of a drug or a biologically active protein, either alone or as a part of a fusionprotein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect,measured ter or teristics of a disease state or condition when administered in one orrepeated doses to a subject. Such effect need not be absolute to be beneficial. Determination of atherapeutically effective amount is well within the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.
] The term “therapeutically effective dose regimen”, as used herein, refers to a schedule forconsecutively administered multiple doses (i.e., at least two or more) of a biologically active protein,either alone or as a part of a fusion protein composition, n the doses are given in therapeuticallyive amounts to result in sustained ial effect on any symptom, aspect, measured parameter orcharacteristics of a disease state or condition.
I). GENERAL TECHNIQUES The practice of the present invention employs, unless otherwise indicated, conventionaltechniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology,genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al.,“Molecular Cloning: A Laboratory Manual,” 3ml edition, Cold Spring Harbor Laboratory Press, 2001;“Current protocols in molecular biology”, F. M. Ausubel, et al. eds.,1987; the series “Methods inEnzymology,” Academic Press, San Diego, CA.; “PCR 2: a practical approach”, M.J. MacPherson, B.D.
Hames and GR. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow,E. and Lane, D. eds., Cold Spring Harbor Laboratory,1988; “Goodman & Gilman’s The PharmacologicalBasis of Therapeutics,” 11th Edition, McGraw—Hill, 2005; and Freshney, R.I., “Culture of Animal Cells:A Manual of Basic Technique,” 4th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents ofwhich are incorporated in their ty herein by reference.
II). COAGULATION FACTOR VIII The present invention s, in part, to compositions comprising factor VIII coagulation factor(CF) linked to one or more extended recombinant proteins (XTEN), resulting in a CFXTEN fusionprotein composition. As used herein, “CF” refers to factor VIII ) or mimetics, sequence tsand truncated versions of FVIII, as described below.
“Factor VIII” or “FVIII” or “FVIII protein” means a blood coagulation factor protein andspecies (including human, porcine, canine, rat or murine FVIII proteins) and sequence variants thereofthat includes, but is not limited to the 2351 amino acid single-chain sor protein (with a 19-aminoacid hobic signal peptide), the mature 2332 amino acid factor VIII cofactor protein ofapproximately 270-330 kDa with the domain structure B-A3-C1-C2, as well as the nonenzymatic“active” or or form of FVIII (FVIIIa) that is a circulating heterodimer of two chains that form as aresult of proteolytic ge after R1648 of a heavy chain form composed of A1 -A2-B (in the range of90-220 kD) of amino acids 1-1648 (numbered ve to the mature FVIII form) and a light chain A3-C1-C2 of 80 kDa of amino acids 1649-2232, each of which is depicted schematically in Further,and as used herein, each of A1, A2 and the A3 domain encompasses acidic spacer regions; a1, a2, and a3acidic regions, respectively. Thus, it will be tood that CFXTEN constructs described as havingA1, A2, A3, B, C1 and C2 domains include the al, a2 and a3 acidic regions. As used herein, “FactorVIII” or “FVIII” or “FVIII polypeptide” also includes variant forms, including proteins with tutions,additions and/or deletions so long as the variant s a desired biological activity such as procoagulantactivity. Myriad onal FVIII variants have been ucted and can be used as recombinant FVIIIproteins as described herein. See PCT Publication Nos. A2, A2, A2, or A2, all of which are incorporated herein by reference in theirentireties. A great many functional FVIII variants are known. In addition, hundreds of nonfunctionalmutations in FVIII have been identified in hemophilia patients. See, e.g., Cutler et al., Hum. Mutat.19:274-8 (2002), incorporated herein by reference in its entirety. In addition, comparisons betweenFVIII from humans and other species have identified conserved residues that are likely to be required forfunction. See, e. g., Cameron et al., Thromb. Haemost. 79:317-22 (1998) and US 632, incorporatedherein by reference in their entireties.
] In one embodiment, the human factor VIII domains are defined by the following amino acidresidues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; B, residues Ser741-Arg1648; A3,residues Ser1649-Asn2019; C1, residues Lys2020-Asn2172; C2, es Ser2l73-Tyr2332. The A3-C1-C2 sequence includes residues Ser1649-Tyr2332. In another embodiment, residues Arg336-Arg372 isusually referred to as the al , and the Arg3 72 is cleaved by in. In certain embodiments, thea2 region is part of the A1 domain. In another embodiment, residues Glul649-Arg1689, is referred to asthe a3 acidic region. In n embodiments, the a3 acidic region is a part of the A3 domain. In anotherembodiment, a native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1,A2, and A3 are the structurally-related ”A domains," B is the ”B domain,” C1 and C2 are the structurally-related ”C domains," and a1, a2 and a3 are acidic spacer s. In the foregoing formula and referringto the primary amino acid sequence position in , the A1 domain of human FVIII s fromAlal to about Arg336, the al spacer region s from about Met337 to about Arg372, the A2 domainextends from about Ser3 73 to about Tyr7l9, the a2 spacer region extends from about Glu720 to aboutArg740, the B domain extends from about Ser741 to about Arg 1648, the a3 spacer region extends fromabout Glul649 to about Arg1689, the A3 domain extends from about Ser1690 to about Asn2019, the C1domain extends from about Lys2020 to about Asn2172, and the C2 domain extends from about Ser2l73to Tyr2332 (Saenko et al., 2005, J Thromb Hemostasis, 1, 922-930). Other than specific proteolyticcleavage sites, designation of the locations of the boundaries between the domains and s of FVIIIcan vary in different literature nces. The boundaries noted herein are therefore designated asapproximate by use of the term “about.” Such factor VIII e truncated sequences such as B-domain deleted “BDD” sequences inwhich a portion or the majority of the B domain sequence is deleted (such as BDD sequences disclosedor referenced in US Pat Nos. 6,818,439 and 7,632,921). An example of a BDD FVIII is REFACTO® orXYNTHA® binant BDD FVIII), which comprises a first polypeptide corresponding to aminoacids 1 to 743 of , fused to a second polypeptide ponding to amino acids 1638 to 2332 of. Exemplary BDD FVIII constructs which can be used to produce recombinant proteins of theinvention include, but are not limited to FVIII with a deletion of amino acids corresponding to aminoacids 747-1638 of mature human FVIII () (Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by nce in its entirety), and FVIII with a on of amino acidscorresponding to amino acids 771—1666 or amino acids 868-1562 of mature human FVIII ()(Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety).
In addition, sequences that e heterologous amino acid insertions or substitutions (such asaspartic acid substituted for valine at on 75), or single chain FVIII (scFVIII) in which the heavy andlight chains are covalently connected by a linker. As used herein, “FVIII” shall be any functional formof factor VIII molecule with the typical characteristics of blood coagulation factor VIII capable ofcorrecting human factor VIII deficiencies when administered to such a subject, e.g., a t withhemophilia A. FVIII or sequence variants have been isolated, characterized, and cloned, as described inUS. Patent or Application Nos. 4,757,006; 4,965,199; 5,004,804; 5,198,349, 5,250,421; 5,919,766;6,228,620; 6,818,439; 7,138,505; 7,632,921; and 20100081615.
Human factor VIII is encoded by a single-copy gene residing at the tip of the long arm of the Xchromosome (q28). It comprises nearly 186,000 base pairs (bp) and tutes approximately 0.1% ofthe X-chromosome (White, G.C. and Shoemaker, C.B., Blood (1989) 73:1-12). The human FVIII aminoacid sequence was d from cDNA as shown in US. Pat. No. 4,965,199, which is incorporatedherein by reference in its entirety. Native mature human FVIII derived from the cDNA sequence (i.e.,t the secretory signal peptide but prior to other post-translational sing) is presented as FIG.
The DNA encoding the mature factor VIII mRNA is found in 26 separate exons ranging in sizefrom 69 to 3,106 bp. The 25 intervening intron regions that separate the exons range in size from 207 to32,400 bp. The complete gene consists of approximately 9 kb of exon and 177 kb of intron. The threerepeat A domains have approximately 30% sequence homology. The B domain contains 19 of theapproximately 25 predicted glycosylation sites, and the A3 domain is believed to contain a g sitefor the von Willebrand factor. The tandem C domains follow the A3 domain and have approximately37% homology to each other (White, G.C. and Shoemaker, C.B., Blood (1989) 73:1-12).
The B domain separates the A2 and A3 domains of native factor FVIII in the newlysynthesized precursor -chain molecule. The precise boundaries of the B domain have beenvariously reported as extending from amino acids 712 to 1648 of the sor sequence (Wood et al.,Nature (1984) 312:330-337) or amino acids 741—1648 (Pipe, SW, Haemophilia (2009) 7—1196and US Pat. No. 7,560,107) or amino acids 740-1689 (Toole, JJ. Proc. Natl. Acad. Sci. USA (1986)9-5942). As used herein, ”B domain” means amino acids 741-1648 of mature factor VIII. Asused herein, “FVIII B domain deletion” or “FVIII BDD” means a FVIII ce with any, a ntof, or all of amino acids 741 to 1648 deleted. In one embodiment, FVIII BDD variants retain remnantamino acids of the B domain from the N-terminal end (“B1” as used herein) and C-terminal end (“B2” asused herein). In one FVIII BDD variant, the B domain remnant amino acids are SFSQNPPVLKRHQR(SEQ ID NO: 1614). In one FVIII BDD variant, the B1 remnant is SFS and the B2 remnant isQNPPVLKRHQR (SEQ ID NO: 1615). In another FVIII BDD variant, the B1 t is SFSQN (SEQID NO: 1616) and the B2 remnant is HQR (SEQ ID NO: 1617). A ain—deleted factorVIII,” ”FVIII BDD,” or ”BDD FVIII” may have the full or partial deletions disclosed in US. Pat. Nos.6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 720,,543,502, 278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each ofwhich is incorporatedherein by reference in its entirety. In some embodiments, a B-domain—deleted factor VIII sequence of thepresent invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 andexamples 1-5 of US. Pat. No. 6,316,226 (also in US 6,346,513). In another embodiment, a B-domaindeleted factor VIII is the S743/Q1638 B-domain deleted factor VIII (SQ version factor VIII) (e. g., factorVIII having a deletion from amino acid 744 to amino acid 1637, e. g., factor VIII having amino acids 1-743 and amino acids 163 8-2332 of full-length factor VIII). In some embodiments, a B-domain-deletedfactor VIII of the present invention has a deletion sed at col. 2, lines 26-51 and examples 5-8 ofUS. Patent No. 5,789,203 (also US 447, US 886, and US 6,228,620). In some embodiments,a B-domain-deleted factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US PatentNo. 5,972,885; col. 6, lines 1-22 and example 1 of US. Patent no. 6,048,720; col. 2, lines 17-46 of US.
Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of US. Patent no. 5,171,844; col. 2, lines 55-68,figure 2, and example 1 ofU.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 ofU.S.
Patent No. 112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col.8, line 26, and col. 11, line 5 to col. 13, line 39 ofU.S. Patent no. 7,041,635; or col. 4, lines 25-53, ofUS. Patent No. 6,458,563. In some ments, a B-domain-deleted factor VIII has a deletion of mostof the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivoproteolytic processing of the primary translation product into two polypeptide chain, as disclosed in W091/09122, which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted factor VIII is constructed with a deletion of amino acids 747-163 8, i. e., lly a completedeletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporatedherein by reference in its ty. A B-domain-deleted factor VIII may also contain a deletion of aminoacids 771—1666 or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6, incorporated herein by reference in its entirety. Additional B domain deletions that are part ofthe invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc.
Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986):8343-8347)), 741 through 1646 (Kaufman (PCT hed application No. WO 87/04187)), 747-1560(Sarver, et al., DNA (1987) 6:553-564)), 741 though 1648 (Pasek (PCT ation No.88/00831)), or816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 25, EP 295597)),each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions may bemade in any factor VIII sequence utilized in the embodiments of the present invention. ns involved in ng e factor I, factor II, factor III, factor IV, factor V, factor VI,factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, Protein C, and tissue factorctively or individually “clotting protein(s)”). The interaction of the major clotting proteins in theintrinsic and extrinsic ng pathways is showed in The majority of the clotting proteins arepresent in zymogen form, but when activated, exhibit a procoagulant protease actiVity in which theyactivate another of the clotting proteins, contributing to the intrinsic or extrinsic coagulation pathway andclot formation. In the intrinsic pathway of the coagulation cascade, FVIII associates with a complex ofactivated factor IX, factor X, calcium, and phospholipid. The factor VIII heterodimer has no enzymaticactivity, but the heterodimer becomes active as a cofactor of the enzyme factor IXa after proteolyticactivation by thrombin or factor Xa, with the actiVity of factor VIIIa characterized by its ability to form ane binding site for factors IXa and X in a conformation suitable for activation of the factor X byfactor IXa. Upon cleavage by thrombin, activated FVIII (FVIIIa) dissociates from von Willebrand factorand binds to negatively charged phospholipid PL, and the resulting complex participates as a cofactor tofactor IXa in the factor X activating (tenase) complex. Within the C2 domain and amino acid residuesWO 22617 1649 through 1689 in the A3 domain are von Willebrand factor (VWF) binding sites that act to complexwith von Willebrand factor, the resulting ating complex protects FVIII from rapid degradation inthe blood (Weiss HJ, et al. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies onposttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest(1977) 60390).
Activated factor VIII is a heterotrimer comprised of the A1 domain and the A2 domain and thelight chain including domains A3-C1-C2. The activation of factor IX is achieved by a two-step removalof the activation peptide (Ala 146-Arg 180) from the molecule (Bajaj et al., Human factor IX and factorIXa, in METHODS IN ENZYMOLOGY. 1993). The first ge is made at the Arg 145-Ala 146 siteby either factor XIa or factor VIIa/tissue . The second, and rate limiting cleavage is made at Arg180-Val 181. The activation removes 35 residues. Activated human factor IX exists as a heterodimer ofthe C-terminal heavy chain (28 kDa) and an N—terminal light chain (18 kDa), which are held together byone disulfide bridge attaching the enzyme to the Gla domain. Factor IXa in turn activates factor X inconcert with activated factor VIII. Alternatively, s IX and X can both be activated by factor VIIacomplexed with lipidated tissue factor, generated Via the extrinsic pathway. Factor Xa then participatesin the final common pathway whereby ombin is converted to thrombin, and in, in turnconverts fibrinogen to fibrin to form the clot.
Defects in the coagulation process can lead to bleeding disorders (coagulopathies) in which thetime taken for clot formation is prolonged. Such s can be congenital or acquired. For example,hemophilia A and B are inherited diseases characterized by deficiencies in FVIII and FIX, respectively.
Stated differently, ically active factor VIII corrects the coagulation defect in plasma derived fromindividuals afflicted with hemophilia A. Recombinant FVIII has been shown to be effective and hasbeen approved for the ent of hemophilia A in adult and pediatric patients, and also is used to stopbleeding episodes or t bleeding associated with trauma and/or surgery. Current therapeutic uses offactor VIII can be problematic in the treatment of indiViduals exhibiting a deficiency in factor VIII, aswell as those indiViduals with Von rand's disease. In addition, individuals receiving factor VIII inement therapy ntly develop antibodies to these proteins that often reduce or eliminate theprocoagulant actiVity of the bound FVIII. Continuing treatment is exceedingly difficult because of thepresence of these antibodies that reduce or negate the efficacy of the treatment.
In one aspect, the invention plates inclusion of FVIII sequences in the CFXTEN fusionprotein compositions that are identical to human FVIII, sequences that have homology to FVIIIsequences, sequences that are natural, such as from humans, non-human primates, mammals (includingdomestic animals), or truncated version of FVIII; all of which retain at least a portion of the procoagulantactiVity of native FVIII and that are useful for preventing, treating, mediating, or ameliorating hemophiliaA or bleeding es d to trauma, surgery, or deficiency of ation factor VIII. Sequenceswith homology to FVIII may be found by standard homology searching techniques, such as NCBIBLAST, or in public databases such as Chemical Abstracts es Databases (e. g., the CAS Registry),GenBank, The Universal Protein Resource (UniProt) and subscription ed databases such asGenSeq (e. g., Derwent).
In one embodiment, the FVIII incorporated into the subject CFXTEN compositions is arecombinant ptide with a sequence corresponding to a FVIII protein found in nature. In anotherembodiment, the FVIII is a tural FVIII sequence variant, fragment, g, or a mimetic of al sequence that retains at least a portion of the procoagulant activity of the corresponding nativeFVIII. In another embodiment, the FVIII is a truncated variant with all or a portion of the B domaindeleted I BDD”), which can be in either heterodimeric form or can remain as a single chain(“scFVIII”), the latter described in Meulien et al., Protein Eng. (1988) 2(4):301-306. Non-limitingexamples of FVIII BDD are factor VIII sequences in which the amino acids are deleted between residuenumber 741 and residue number 1640 (numbered relative to native, mature FVIII), or n residuenumber 745 and residue number 1640, or between e number 745 and residue number 1640, orn residue number 741 and residue number 1690, or between residue number 745 and residuenumber 1667, or between residue number 745 and residue number 1657, or between residue number 747and residue number 1642, or between residue number 751 and residue number 1667.
In another embodiment, heterologous sequences are incorporated into the FVIII, which maye XTEN, as described more fully below. Table 1 provides a non-limiting list of amino acidsequences of FVIII that are encompassed by the CFXTEN fusion proteins of the invention. In someembodiments, FVIII incorporated into CFXTEN fusion proteins e proteins that have at least about70% sequence identity, or alternatively 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an aminoacid sequence of able length selected from Table 1.
Table 1: FVIII amino acid seguencesNameAmino Acid Sequence ID(source)FVIII MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 1precursor SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMpolypeptide ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN(human) GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEWO 22617 NameAmino Acid Sequence ID(source)GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 2mature DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS(human) EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGNameAmino Acid Sequence ID(source)QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII MQVELYTCCFLCLLPFSLSATRKYYLGAVELSWDYMQSDLLSALHADTSFSSRVP 3(Canine) GSLPLTTSVTYRKTVFVEFTDDLFNIAKPRPPWMGLLGPTIQAEVYDTVVIVLKNMASHPVSLHAVGVSYWKASEGAEYEDQTSQKEKEDDNVIPGESHTYVWQVLKENGPMASDPPCLTYSYFSHVDLVKDLNSGLIGALLVCKEGSLAKERTQTLQEFVLLGKSWHSETNASLTQAEAQHELHTINGYVNRSLPGLTVCHKRSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTFLMDLGQFLLFCHIPSHQHDGMEAYVKVDSCPEEPQLRMKNNEDKDYDDGLYDSDMDVVSFDDDSSSPFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPSGPTPNDRSHKNLYLNNGPQRIGKKYKKVRFVAYTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGINYVTPLHTGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRSWYLTENMQRFLPNADVVQPHDPEFQLSNIMHSINGYVFDNLQLSVCLHEVAYWYILSVGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFRNRGMTALLKVSSCNRNIDDYYEDTYEDIPTPLLNENNVIKPRSFSQNSRHPSTKEKQLKATTTPENDIEKIDLQSGERTQLIKAQSVSSSDLLMLLGQNPTPRGLFLSDLREAHSRGAIERNKGPPEVASLRPELRHSEDREFTPEPELQLRLNENLGTNTTVELKKLDLKISSSSDSLMTSPTIPSDKLAAATEKTGSLGPPNMSVHFNSHLGTIVFGNNSSHLIQSGVPLELSEEDNDSKLLEAPLMNIQESSLRENVLSMESNRLFKEERIRGPASLIKDNALFKVNISSVKTNRAPVNLTTNRKTRVAIPTLLIENSTSVWQDIMLERNTEFKEVTSLIHNETFMDRNTTALGLNHVSNKTTLSKNVEMAHQKKEDPVPLRAESKIPFLPDWIKTHGKNSLSSEQRPSPKQLTSLGSEKSVKDQNFLSEEKVVVGEDEFTKDTELQEIFPNNKSIFFANLANVQENDTYNQEKKSPEEIERKEKLTQENVALPQAHTMIGTKNFLKNLFLLSTKQNVAGLEEQPYTPILQDTRSLNDSPHSEGIHMANFSKIREEANLEGLGNQTNQMVERFPSTTRMSSNASQHVITQRGKRSLKQPRLSQGEIKFERKVIANDTSTQWSKNMNYLAQGTLTQIEYNEKEKRAITQSPLSDCSMRNHVTIQMNDSALPVAKESASPSVRHTDLTKIPSQHNSSHLPASACNYTFRERTSGVQEGSHFLQEAKRNNLSLAFVTLGITEGQGKFSSLGKSATNQPMYKKLENTVLLQPGLSETSDKVELLSQVHVDQEDSFPTKTSNDSPGHLDLMGKIFLQKTQGPVKMNKTNSPGKVPFLKWATESSEKIPSKLLGVLAWDNHYDTQIPSEEWKSQKKSQTNTAFKRKDTILPLGPCENNDSTAAINEGQDKPQREAMWAKQGEPGRLCSQNPPVSKHHQREITVTTLQPEEDKFEYDDTFSIEMKREDFDIYGDYENQGLRSFQKKTRHYFIAAVERLWDYGMSRSPHILRNRAQSGDVQQFKKVVFQEFTDGSFTQPLYRGELNEHLIRAEVEDNIVVTFKNQASRPYSFYSSLISYDEDEGQGAEPRRKFVNPNETVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRSNTLNPAHGRQVTVQEFALVFTIFDETKSWYFTENLERNCRAPCNVQKEDPTLKENFRFHAINGYVKDTLPGLVMAQDQKVRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETVEMLPSQVGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDLLAPMIIHGIMTQGARQKFSSLYVSQFIIMYSLDGNKWHSYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIAQYIRLHPTHYSIRSTLRMELLGCDFNSCSMPLGMESKAISDAQITASSYLSSMLATWSPSQARLHLQGRTNAWRPQANNPKEWLQVDFRKTMKVTGITTQGVKSLLISMYVKEFLISSSQDGHNWTLFLQNGKVKVFQGNRDSSTPVRNRLEPPLVARYVR LHPQSWAHHIALRLEVLGCDTQQPAFVIII (Pig) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 4DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLNameAmino Acid Sequence ID(source)IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII AIRRYYLGAVELSWNYIQSDLLSVLHTDSRFLPRMSTSFPFNTSIMYKKTVFVEYK 5(Mouse) DQLFNIAKPRPPWMGLLGPTIWTEVHDTVVITLKNMASHPVSLHAVGVSYWKASEGDEYEDQTSQMEKEDDKVFPGESHTYVWQVLKENGPMASDPPCLTYSYMSHVDLVKDLNSGLIGALLVCKEGSLSKERTQMLYQFVLLFAVFDEGKSWHSETNDSYTQSMDSASARDWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEIHSIFLEGHTFFVRNHRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKVDSCPEESQWQKKNNNEEMEDYDDDLYSEMDMFTLDYDSSPFIQIRSVAKKYPKTWIHYISAEEEDWDYAPSVPTSDNGSYKSQYLSNGPHRIGRKYKKVRFIAYTDETFKTRETIQHESGLLGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVSPLHARRLPRGIKHVKDLPIHPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINPERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSIFDENQSWYITENMQRFLPNAAKTQPQDNIMHSINGYVFDSLELTVCLHEVAYWHILSVGAQTDFLSIFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFRKRGMTALLKVSSCDKSTSDYYEEIYEDIPTQLVNENNVIDPRSFFQNTNHPNTRKKKFKDSTIPKNDMEKIEPQFEEIAEMLKVQSVSVSDMLMLLGQSHPTPHGLFLSDGQEAIYEAIHDDHSPNAIDSNEGPSKVTQLRPESHHSEKIVFTPQPGLQLRSNKSLETTIEVKWKKLGLQVSSLPSNLMTTTILSDNLKATFEKTDSSGFPDMPVHSSSKLSTTAFGKKAYSLVGSHVPLNASEENSDSNILDSTLMYSQESLPRDNILSIENDRLLREKRFHGIALLTKDNTLFKDNVSLMKTNKTYNHSTTNEKLHTESPTSIENSTTDLQDAILKVNSEIQEVTALIHDGTLLGKNSTYLRLNHMLNRTTSTKNKDIFHRKDEDPIPQDEENTIMPFSKMLFLSESSNWFKKTNGNNSLNSEQEHSPKQLVYLMFKKYVKNQSFLSEKNKVTVEQDGFTKNIGLKDMAFPHNMSIFLTTLSNVHENGRHNQEKNIQEEIEKEALIEEKVVLPQVHEATGSKNFLKDILILGTRQNISLYEVHVPVLQNITSINNSTNTVQIHMEHFFKRRKDKETNSEGLVNKTREMVKNYPSQKNITTQRSKRALGQFRLSTQWLKTINCSTQCIIKQIDHSKEMKKFITKSSLSDSSVIKSTTQTNSSDSHIVKTSAFPPIDLKRSPFQNKFSHVQASSYIYDFKTKSSRIQESNNFLKETKINNPSLAILPWNMFIDQGKFTSPGKSNTNSVTYKKRENIIFLKPTLPEESGKIELLPQVSIQEEEILPTETSHGSPGHLNLMKEVFLQNameAmino Acid Sequence ID(source)KIQGPTKWNKAKRHGESIKGKTESSKNTRSKLLNHHAWDYHYAAQIPKDMWKSKEKSPEIISIKQEDTILSLRPHGNSHSIGANEKQNWPQRETTWVKQGQTQRTCSQIPPVLKRHQRELSAFQSEQEATDYDDAITIETIEDFDIYSEDIKQGPRSFQQKTRHYFIAAVERLWDYGMSTSHVLRNRYQSDNVPQFKKVVFQEFTDGSFSQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKEDQRGEEPRRNFVKPNETKIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLERDMHSGLIGPLLICHANTLNPAVQEFALLFTIFDETKSWYFTENVKRNCKTPCNFQMEDPTLKENYRFHA1NGYVMDTLPGLVMAQDQRIRWYLLSMGNNENIQSIHFSGHVFTVRKKEEYKMAVYNLYPGVFETLEMIPSRAGIWRVECLIGEHLQAGMSTLFLVYSKQCQIPLGMASGSIRDFQITASGHYGQWAPNLARLHYSGSINAWSTKEPFSWIKVDLLAPMIVHGIKTQGARQKFSSLYISQFIIMYSLDGKKWLSYQGNSTGTLMVFFGNVDSSGIKHNSFNPPIIARYIRLHPTHSSIRSTLRMELMGCDLNSCSIPLGMESKVISDTQITASSYFTNMFATWSPSQARLHLQGRTNAWRPQVNDPKQWLQVDLQKTMKVTGIITQGVKSLFTSMFVKEFLISSSQDGHHWTQILYNGKVKVFQGNQDSSTPMMNSLDPPLLTRYLRIHPQIWEHQIALRLEILGCEAQQQYFVIII BDD MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKvariant SFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM(US Pat ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENNo. GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF763292 1, AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSSEQ ID VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLNO: 3) FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTBDD-Z AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDWO 22617 NameAmino Acid Sequence ID(source)EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 8BDD-3 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS(G1 648) EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALREAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 9BDD-4 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDEAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLCYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYINameAmino Acid Sequence ID(source)RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYSSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 10BDD-5 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLCYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 11BDD-6 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 12BDD-7 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVNameAmino Acid Sequence ID(source)DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 1 3BDD-8 SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMprecursor LHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN(US Pat. PLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFN0. AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS681 8439 VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLSEQ ID FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDNO: 47) DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 14BDD-9 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASmature EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV(US Pat. DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMN0. QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL681 8439) EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPNameAmino Acid Sequence ID(source)KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 1 5BDD- 1 0 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTS V V Y KKTLF 16BDD-1 1 VEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQNameAmino Acid Sequence ID(source)EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 1 7BDD- 12 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 1 8BDD- 1 3 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD(31:32:) Amino Acid Sequence IDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY The present invention also contemplates CFXTEN sing FVIII with various amino aciddeletions, insertions and substitutions made in the FVIII sequences of Table 1 that retain procoagulantactivity. Examples of conservative tutions for amino acids in polypeptide sequences are shown inTable 2. In embodiments of the CFXTEN in which the sequence identity of the FVIII is less than 100%compared to a specific sequence disclosed herein, the invention contemplates substitution of any of theother 19 natural L-amino acids for a given amino acid residue of the given FVIII, which may be at anyposition within the sequence of the FVIII, including adjacent amino acid residues. If any one substitutionresults in an undesirable change in procoagulant activity, then one of the ative amino acids can beemployed and the construct protein evaluated by the methods described herein (e. g., the assays of Table49), or using any of the techniques and guidelines for conservative and non-conservative mutations setforth, for instance, in US. Pat. No. 5,364,934, the content of which is incorporated by reference in itsentirety, or using methods generally known in the art. In a preferred substitution, the FVIII entof the CFXTEN embodiments is modified by replacing the R1648 residue (numbered relative to thenative mature form of FVIII) with glycine or alanine to prevent proteolytic processing to the heterodimerform. In another substitution, the FVIII component of the CFXTEN embodiments is modified byreplacing the Y1680 residue (numbered relative to the native mature form of FVIII) with phenylalanine.
In r embodiment, the FVIII ent of the CFXTEN embodiments is modified by replacing theY1680 residue (numbered ve to the native mature form of FVIII) with phenylalanine and the R1648residue (numbered ve to the native mature form of FVIII) with glycine or alanine.
In one embodiment, the FVIII of the fusion protein composition has one or more amino acidsubstitutions designed to reduce the g of FVIII tors at epitopes recognized by the antibodiesof Table 9, including but not limited to substitutions at Lys(377), Lys(466), Lys(3 80), Ser(488),Arg(489), Arg(490), Leu(491), Lys(493), Lys(496), His(497), Lys(499), Lys(512), Lys(523), Lys(556),Met (2199), Phe(2200), Leu(2252), Val(2223), and Lys(2227). In addition, variants can include, forinstance, polypeptides wherein one or more amino acid residues are added or d at or near the N— orC-terminus of the full-length native amino acid sequence or of a domain of a FVIII so long as the ts some if not all of the procoagulant actiVity of the native peptide. The resulting FVIII sequencesthat retain at least a portion (e. g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least95% or more) of the procoagulant actiVity in comparison to native circulating FVIII are ered usefulfor the fusion n compositions of this invention. Examples of FVIII variants are known in the art,including those described in US Patent and Application Nos. 6,316,226; 6,818,439; 7,632,921;20080227691, which are incorporated herein by reference. In one embodiment, a PV111 sequence varianthas an aspartic acid substituted for valine at amino acid position 75 (numbered relative to the nativemature form of FVIII).
Table 2: Exemplary conservative amino acid substitutionsOriginal e Exemplary SubstitutionsAla (A) val; leu; ileArg (R) lys; gln; asnAsn (N) gin; his; lys; argAsp (D) GluCys (C) SerGln (Q) AsnGlu (E) AspGly (G) ProHis (H) asn: gin: lys; arglle (l) leu; val; met; ala; phe: norleucineLeu (L) norleucine: ile: val; met; ala: pheLys (K) arg: gin: asnMet (M) leu; phe; ilePhe (F) leu: val: ile; alaPro (P) GlySer (S) ThrThr (T) SerTrp (W) TyrTyr(Y) Trp: phe: thr: serVal (V) lle; leu; met; phe; ala; cineIII). EXTENDED RECOMBINANT POLYPEPTIDES In one aspect, the invention provides XTEN polypeptide compositions that are useful as fusionprotein partner(s) to link to and/or incorporate Within a FVIII polypeptide, resulting in a CFXTEN fusionprotein. XTEN are generally polypeptides With turally occurring, substantially non-repetitivesequences having a low degree of or no secondary or tertiary structure under physiologic conditions.
XTEN typically have from about 36 to about 3000 amino acids of Which the majority or the entirety aresmall hydrophilic amino acids. As used , “XTEN” specifically excludes Whole antibodies ordy fragments (e. g. single-chain antibodies and PC fragments). XTEN polypeptides have utility as afusion protein partners in that they serve various roles, conferring certain desirable pharmacokinetic,physicochemical, pharmacologic, and pharmaceutical properties When linked to a FVlll protein to acreate a CFXTEN fusion protein. Such CFXTEN fusion protein itions have ed propertiescompared to the corresponding FVIH not linked to XTEN, making them useful in the treatment of certainconditions d to FVHI encies or bleeding disorders, as more fully described below.
The ion ia for the XTEN to be fused to the FVIII proteins used to create theinventive fusion proteins compositions generally relate to attributes of physical/chemical properties andconformational structure of the XTEN that is, in turn, used to confer enhanced pharmaceutical,pharmacologic, and pharmacokinetic properties to the FVIII fusion proteins compositions. Theunstructured characteristic and al/chemical properties of the XTEN result, in part, from the overallamino acid composition portionately limited to 4-6 hydrophilic amino acids, the linking of theamino acids in a quantifiable non-repetitive design, and the length of the XTEN polypeptide. In anadvantageous feature common to XTEN but uncommon to polypeptides, the ties ofXTENdisclosed herein are not tied to absolute primary amino acid ces, as evidenced by the diversity ofthe exemplary sequences of Table 4 that, within varying ranges of length, s similar properties,many of which are documented in the Examples. The XTEN of the present invention may exhibit one ormore, or all of the following advantageous properties: unstructured conformation, conformationalflexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, lowbinding to mammalian ors, a defined degree of , and increased hydrodynamic (or Stokes)radii; properties that can make them particularly useful as fusion protein partners. Non-limitingexamples of the enhanced properties that XTEN confer on the fusion proteins comprising FVIII fused toXTEN, compared to FVIH not linked to XTEN, include increases in the overall solubility and/ormetabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, reduced rate ofabsorption when administered subcutaneously or intramuscularly, d binding to FVIII clearancereceptors, reduced reactivity to anti-payload antibodies, enhanced interactions with substrate, and/orenhanced pharmacokinetic properties when administered to a subject. The enhanced pharmacokineticproperties of the CFXTEN compositions compared to FVIII not linked to XTEN include longer terminalhalf-life (e. g., two-fold, three-fold, four-fold or more), increased area under the curve (AUC) (e. g., 25%,50%, 100% or more), lower volume of distribution, and enhanced absorption after subcutaneous orintramuscular injection (an advantage compared to commercially-available forms of FVIII that must beadministered intravenously). In addition, it is believed that the CFXTEN compositions comprisingcleavage sequences (described more fully, below) permit sustained release of biologically active FVIH,such that the administered CFXTEN acts as a depot. It is specifically contemplated that the inventiveCFXTEN fusion proteins can exhibit one or more or any combination of the improved propertiesdisclosed herein. As a result of these enhanced properties, it is believed that CFXTEN compositionspermit less frequent dosing compared to FVIH not linked to XTEN when administered at comparables. Such CFXTEN fusion protein itions have utility to treat certain factor VIII-relatedconditions, as described herein.
] A variety of methods and assays are known in the art for determining the physical/chemicalties of proteins such as the CFXTEN compositions comprising XTEN. Such properties include butare not limited to ary or ry ure, solubility, protein aggregation, stability, absolute andapparent molecular , purity and uniformity, melting properties, contamination and water t.
Methods to assay these properties include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism,differential ng metry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR,Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed inArnau, er al., Prot Expr and Purif (2006) 48, 1-13.
The XTEN component(s) of the CFXTEN are designed to behave like denatured peptidesequences under physiological conditions, e the extended length of the polymer. “Denatured”describes the state of a peptide in solution that is characterized by a large conformational freedom of thee backbone. Most peptides and proteins adopt a denatured mation in the presence of highconcentrations of denaturants or at elevated temperature. es in denatured conformation have, forexample, characteristic circular ism (CD) spectra and are characterized by a lack of long-rangections as determined by NMR. “Denatured conformation” and “unstructured conformation” areused synonymously herein. In some embodiments, the invention provides XTEN sequences that, underphysiologic conditions, are largely devoid of secondary structure. In other cases, the XTEN sequencesare substantially devoid of secondary structure under physiologic conditions such that the XTEN canadopt random coil conformation. “Largely devoid,” as used in this context, means that at least 50% ofthe XTEN amino acid residues of the XTEN sequence do not contribute to ary structure asmeasured or determined by the means described herein. “Substantially devoid,” as used in this context,means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at leastabout 99% of the XTEN amino acid residues of the XTEN sequence do not bute to secondarystructure, as measured or determined by the methods described herein.
A variety of methods have been ished in the art to discern the presence or absence ofsecondary and tertiary structures in a given ptide. In particular, secondary structure can bemeasured spectrophotometrically, e. g., by circular dichroism spectroscopy in the “far-UV” alregion (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise toa characteristic shape and ude of CD spectra, as does the lack of these structure elements.
Secondary structure can also be predicted for a polypeptide sequence via certain computer programs oralgorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13:222-45) and the -Osguthorpe-Robson (“GOR”) algorithm (Gamier J, Gibrat JF, Robson B.
, GOR method for predicting protein secondary structure from amino acid ce. sEnzym01266:540-553), as described in US Patent Application Publication No. 20030228309A1. For agiven sequence, the algorithms can predict Whether there exists some or no secondary structure at all,expressed as the total and/or percentage of residues of the sequence that form, for example, alpha-helicesor heets or the percentage of residues of the sequence predicted to result in random coil formation(which lacks secondary structure).
In one embodiment, the XTEN ces used in the subject fusion protein compositions havean alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou-Fasmanalgorithm. In another embodiment, the XTEN sequences of the fusion protein compositions have a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm.
In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helixpercentage ranging from 0% to less than about 5% and a beta-sheet tage ranging from 0% to lessthan about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTENsequences of the fusion protein compositions have an alpha-helix tage less than about 2% and abeta-sheet percentage less than about 2%. The XTEN sequences of the fusion n compositions havea high degree of random coil percentage, as determined by the GOR algorithm. In some embodiments,an XTEN sequence have at least about 80%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, and most preferably at least about 99% random coil, as determined by the GORalgorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-heliX percentage ranging from 0% to less than about 5% and a beta-sheet tage ranging from 0% toless than about 5% as determined by the Chou-Fasman algorithm and at least about 90% random coil, asdetermined by the GOR algorithm. In other embodiments, the XTEN sequences of the fiJsion ncompositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less thanabout 2% at least about 90% random coil, as determined by the GOR algorithm.1. Non-repetitive Sequences It is contemplated that the XTEN sequences of the CFXTEN embodiments are substantiallynon-repetitive. In l, repetitive amino acid sequences have a tendency to aggregate or form higherorder structures, as exemplified by natural repetitive sequences such as collagens and leucine zippers.
These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystalinestructures. In contrast, the low tendency of non-repetitive sequences to aggregate enables the design oflong-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise belikely to aggregate if the sequences were repetitive. The non-repetitiveness of a subject XTEN can beed by assessing one or more of the ing features. In one embodiment, a “substantially non-repetitive” XTEN ce has about 36, or at least 72, or at least 96, or at least 144, or at least 288, or atleast 400, or at least 500, or at least 600, or at least 700, or at least 800, or at least 864, or at least 900, orat least 1000, or at least 2000, to about 3000 or more amino acid residues, or has a length ranging fromabout 36 to about 3000, about 100 to about 500, about 500 to about 1000, about 1000 to about 3000amino acids and residues, in which no three contiguous amino acids in the sequence are identical aminoacid types unless the amino acid is serine, in which case no more than three contiguous amino acids areserine residues. In r embodiment, as described more fully below, a “substantially non-repetitive”XTEN sequence comprises motifs of 9 to 14 amino acid es wherein the motifs consist of 4 to 6types of amino acids ed from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is notrepeated more than twice in the sequence motif.
The degree of repetitiveness of a ptide or a gene can be measured by computer msor algorithms or by other means known in the art. According to the current invention, algorithms to beused in calculating the degree of repetitiveness of a particular polypeptide, such as an XTEN, areWO 22617 disclosed herein, and examples of sequences analyzed by algorithms are provided (see Examples, below).
In one , the repetitiveness of a polypeptide of a predetermined length can be calculated (hereinafterquence score”) according to the a given by Equation 1:Subsequence score Em}. C $33.31: g‘ , . Iwherein: m = (amino acid length of polypeptide) — (amino acid length of uence) +1; and Count,- = cumulative number of occurrences of each unique subsequence withinsequence,- An algorithm termed “SegScore” was developed to apply the foregoing equation to quantitaterepetitiveness of polypeptides, such as an XTEN, providing the subsequence score wherein sequences ofa predetermined amino acid length “n” are analyzed for repetitiveness by determining the number oftimes (a “count”) a unique subsequence of length “s” appears in the set length, d by the absolutenumber of subsequences within the predetermined length of the sequence. depicts a logicflowchart of the SegScore algorithm, while portrays a schematic of how a subsequence score isderived for a fictitious XTEN with 11 amino acids and a subsequence length of 3 amino acid residues.
For example, a ermined polypeptide length of 200 amino acid residues has 192 overlapping 9-amino acid subsequences and 198 3-mer uences, but the subsequence score of any givenpolypeptide will depend on the absolute number of unique subsequences and how frequently each uniquesubsequence (meaning a different amino acid sequence) appears in the predetermined length of thesequence.
In the t of the t invention, “subsequence score” means the sum of ences ofeach unique 3-mer frame across 200 consecutive amino acids of the cumulative XTEN polypeptidedivided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence.
Examples of such subsequence scores derived from 200 consecutive amino acids of tive and nonrepetitivepolypeptides are presented in e 45. In one embodiment, the invention es aCFXTEN comprising one XTEN in which the XTEN has a subsequence score less than 12, morepreferably less than 10, more preferably less than 9, more preferably less than 8, more preferably lessthan 7, more preferably less than 6, and most preferably less than 5. In another embodiment, theinvention provides CFXTEN comprising at least two to about six XTEN in which 200 amino acids of theXTEN have a subsequence score of less than 10, more preferably less than 9, more preferably less than 8,more preferably less than 7, more preferably less than 6, and most preferably less than 5. In theembodiments of the CFXTEN fusion protein compositions described herein, an XTEN component of afusion protein with a subsequence score of 10 or less (i.e., 9, 8, 7, etc.) is also substantially non-repetitive.
It is believed that the non-repetitive characteristic ofXTEN of the present invention erwith the particular types of amino acids that predominate in the XTEN, rather than the absolute primarysequence, confers many of the enhanced physicochemical and biological properties of the CFXTENfusion proteins. These enhanced properties include a higher degree of expression of the fusion protein inthe host cell, greater genetic stability of the gene encoding XTEN, a r degree of solubility, lesscy to aggregate, and enhanced pharmacokinetics of the resulting CFXTEN compared to fusionproteins comprising polypeptides having repetitive sequences. These enhanced properties permit moreefficient manufacturing, lower cost of goods, and facilitate the formulation of XTEN-comprisingpharmaceutical preparations containing extremely high protein concentrations, in some cases ing100 mg/ml. Furthermore, the XTEN polypeptide sequences of the embodiments are designed to have alow degree of internal repetitiveness in order to reduce or substantially eliminate immunogenicity whenadministered to a mammal. Polypeptide sequences composed of short, repeated motifs largely limited toonly three amino acids, such as glycine, serine and glutamate, may result in relatively high antibody titerswhen administered to a mammal despite the absence of predicted T-cell epitopes in these sequences.
This may be caused by the repetitive nature of polypeptides, as it has been shown that immunogens withrepeated epitopes, including protein aggregates, cross-linked immunogens, and repetitive carbohydratesare highly genic and can, for example, result in the cross-linking of B-cell receptors g B-cell activation. sson, J., et al. (2007) e, 25 82 ; Yankai, Z., et al. (2006) Biochems Res Commun, 345 :1365-71 ; Hsu, C. T., et al. (2000) Cancer Res, 1-5); Bachmann MF,et al. Eur J Immunol. (1995) 25(12):3445-3451).2. Exemplary Sequence Motifs The t invention encompasses XTEN used as fusion partners that comprise multiple unitsof shorter sequences, or motifs, in which the amino acid sequences of the motifs are petitive. Thenon-repetitive property is met despite the use of a “building block” approach using a library of sequencemotifs that are multimerized to create the XTEN sequences. Thus, while an XTEN sequence may consistof multiple units of as few as four ent types of sequence motifs, e the motifs themselvesgenerally consist of non-repetitive amino acid sequences, the overall XTEN sequence is designed torender the sequence substantially non-repetitive.
In one embodiment, an XTEN has a substantially non-repetitive sequence of greater than about36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues, or evenlonger wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, orat least about 97%, or about 100% of the XTEN sequence consists of erlapping ce motifs,and wherein each of the motifs has about 9 to 36 amino acid residues. In other ments, at leastabout 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, orabout 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of themotifs has 9 to 14 amino acid residues. In still other embodiments, at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTENsequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acidresidues. In these embodiments, it is preferred that the sequence motifs are composed of substantially(e. g., 90% or more) or exclusively small hydrophilic amino acids, such that the overall sequence has anunstructured, e characteristic. Examples of amino acids that are included in XTEN are, e. g.,arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. As aresult of testing variables such as codon optimization, assembly polynucleotides encoding sequencemotifs, expression of n, charge distribution and solubility of expressed protein, and secondary andry structure, it was discovered that XTEN compositions with the ed characteristics disclosedherein mainly or exclusively include glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P) residues wherein the sequences are designed to be substantially non-repetitive. In oneembodiment, XTEN sequences have predominately four to six types of amino acids selected fromglycine (G), e (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in asubstantially non-repetitive sequence that is greater than about 36 to about 3000, or about 100 to about2000, or about 144 to about 1000 amino acid residues in length. In some embodiment, an XTENsequence is made of 4, 5, or 6 types of amino acids selected from the group consisting of glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, XTEN havesequences of greater than about 36 to about 1000, or about 100 to about 2000, or about 400 to about 3000amino acid residues n at least about 80% of the sequence ts of non-overlapping sequencemotifs wherein each of the motifs has 9 to 36 amino acid es and wherein at least 90%, or at least91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or100% of each of the motifs consists of 4 to 6 types of amino acids selected from glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acidtype in the full-length XTEN does not exceed 30%. In other embodiments, at least about 90% of theXTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G),e (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any oneamino acid type in the full-length XTEN does not exceed 40%, or about 30%, or 25%, or about 17%. Inother embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequencemotifs wherein each of the motifs has 12 amino acid residues consisting of 4 to 6 types of amino acidsselected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), andwherein the content of any one amino acid type in the ength XTEN does not exceed 40%, or 30%,or about 25%. In yet other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%,or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% ofthe XTEN sequence consists of non-overlapping ce motifs wherein each of the motifs has 12amino acid residues consisting of glycine (G), alanine (A), serine (S), threonine (T), ate (E) ande (P).
In still other embodiments, XTENs comprise substantially non-repetitive sequences of greaterthan about 36 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% of the sequence consists of erlapping ce motifs of 9 to 14 aminoacid residues wherein the motifs t of 4 to 6 types of amino acids selected from glycine (G), alanine(A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any twocontiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.
In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists ofnon-overlapping sequence motifs of 12 amino acid residues n the motifs consist of four to sixtypes of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequencemotif is not repeated more than twice in the sequence motif. In other embodiments, at least about 90%,or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, orabout 98%, or about 99% of an XTEN sequence ts of non-overlapping sequence motifs of 12amino acid es wherein the motifs consist of glycine (G), alanine (A), serine (S), ine (T),glutamate (E) and e (P), and wherein the sequence of any two contiguous amino acid residues inany one sequence motif is not repeated more than twice in the ce motif In yet otherembodiments, XTENs consist of 12 amino acid ce motifs wherein the amino acids are selectedfrom glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein thesequence of any two contiguous amino acid residues in any one sequence motif is not repeated more thantwice in the sequence motif, and wherein the content of any one amino acid type in the ength XTENdoes not exceed 30%. The foregoing ments are examples of substantially non-repetitive XTENsequences. onal examples are detailed below.
] In some embodiments, the invention provides CFXTEN compositions comprising one, or two,or three, or four, five, six or more non-repetitive XTEN sequence(s) of about 36 to about 1000 aminoacid es, or cumulatively about 100 to about 3000 amino acid residues wherein at least about 80%,or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multipleunits of four or more non-overlapping sequence motifs selected from the amino acid sequences of Table3, wherein the overall sequence remains substantially non-repetitive. In some embodiments, the XTENcomprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from a single motif family selected from Table 3, resulting in a familysequence. As used herein, y” means that the XTEN has motifs selected only from a single motifcategory from Table 3; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other aminoacids in the XTEN not from a family motif are selected to achieve a needed property, such as to permitincorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or toachieve a better linkage to a FVIII coagulation factor component of the CFXTEN. In some embodimentsofXTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs ofthe AD motif family, or of the AE motif family, or of the AF motif family, or of the AG motif family, orof the AM motif family, or of the AQ motif family, or of the BC family, or of the BD family, with theing XTEN exhibiting the range of homology described above. In other embodiments, the XTENcomprises multiple units of motif sequences from two or more of the motif families of Table 3. Thesesequences can be selected to achieve desired physical/chemical characteristics, including such propertiesas net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred bythe amino acid composition of the motifs, described more fully below. In the embodiments hereinabovedescribed in this paragraph, the motifs incorporated into the XTEN can be selected and assembled usingthe s described herein to e an XTEN of about 36 to about 3000 amino acid residues.
Table 3: XTEN Seguence Motifs of 12 Amino Acids and Motif FamiliesAD GESPGGSSGSES 19AD GSEGSSGPGESS 20AD GSSESGSSEGGP 21AD GSGGEPSESGSS 22AE, AM GSPAGSPTSTEE 23AE, AM, AQ GSEPATSGSETP 24AE, AM, AQ GTSESATPESGP 25AE, AM, AQ GTSTEPSEGSAP 26AF, AM GSTSESPSGTAP 27AF, AM GTSTPESGSASP 28AF, AM ESSTAP 29AF, AM GSTSSTAESPGP 30AG, AM GTPGSGTASSSP 31AG, AM GSSTPSGATGSP 32AG, AM GSSPSASTGTGP 33AG, AM GASPGTSSTGSP 34AQ GEPAGSPTSTSE 35AQ GTGEPSSTPASE 36AQ GSGPSTESAPTE 37AQ GSETPSGPSETA 38AQ GPSETSTSEPGA 39AQ GSPSEPTEGTSA 40BC GSGASEPTSTEP 41BC GSEPATSGTEPS 42BC GTSEPSTSEPGA 43BC SEPGSA 44BD GSTAGSETSTEA 45BD GSETATSGSETA 46BD GTSESATSESGA 47BD GTSTEASEGSAS 48permutations, results in a “family sequence”] In some embodiments ofXTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, the AE motif family, or the AF motif family, or theAG motif family, or the AM motif family, or the AQ motif , or the BC , or the BD family,with the resulting XTEN ting the range of homology bed above. In other embodiments, theXTEN comprises multiple units of motif sequences from two or more of the motif families of Table 3,selected to achieve desired physicochemical characteristics, including such properties as net charge, lackof secondary structure, or lack of repetitiveness that may be conferred by the amino acid composition ofthe motifs, described more fully below. In the embodiments hereinabove described in this paragraph, themotifs or portions of the motifs incorporated into the XTEN can be selected and assembled using themethods described herein to achieve an XTEN of about 36, about 42, about 72, about 144, about 288,about 576, about 864, about 1000, about 2000 to about 3000 amino acid residues, or any intermediatelength. Non-limiting examples ofXTEN family sequences useful for incorporation into the subjectCFXTEN are presented in Table 4. It is intended that a specified ce mentioned relative to Table 4has that sequence set forth in Table 4, While a generalized reference to an AEl44 sequence, for example,is intended to encompass any AE sequence having 144 amino acid residues; e. g., 1A,AEl44_2A, etc., or a generalized reference to an AG144 sequence, for example, is intended toencompass any AG sequence having 144 amino acid residues, e. g., AG144_1, AG144_2, AG144_A,AG144_B, AG144_C, etc.
Table 4: XTEN PolypeptidesXTENAmino Acid Sequence IDQNameAE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS 49AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 50AE42_2 PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG 51AE42_3 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP 52AG42_1 GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP 53AG42_2 GPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP 54AG42_3 SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA 55AG42_4 SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG 56AE48 SPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS 57AM48 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS 58AE 1 44 SGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS 59SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPAEl44_ SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS 601A TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAEl44_ TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS 612A TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESPGTSESATPESGPGAEl44_ TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS 622B TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESPGTSESATPESGPGAEl44_ SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS 633A TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGEGTSTEPSEGSAPGAE 1 44_ SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS 643B TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGAEl44_ TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 654A TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAEl44_ TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 664B TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGWO 22617 XTENAmino Acid Sequence IDNameAE144_ TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 675A TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGAE144_ TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE 686B PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGAF144 GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGS 69TSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPAG144_ SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA 701 STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPAG144_ PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 712 GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSAG144_ GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 72A SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPAG144_ GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG 73B SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPAG144_ TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPG 74C TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPAG144_ GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG 75F TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPAG144_ GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG 763 ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPAG144_ GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG 774 ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPAE288_ GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 781 STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAE288_ GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT 79STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPAG288_ PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP 80GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSAG288_ GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG 81ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPAF504 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 82SXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTXTENAmino Acid Sequence IDNamePGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPAF54O GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT 83SASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSEPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPAD576 GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPG 84SSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSSGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESSAE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 85STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPAF576 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT 86STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPAGS76 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSP 87GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSAE624 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 88GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPXTENAmino Acid Sequence IDNameGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPAD836 GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESG 89ESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSEPSESGSSGESPGGSSGSESGSGGEPSESGSSAE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 90STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAF864 GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT 91STPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPAG864_ GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 92TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAXTENAmino Acid Sequence IDNameSTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTAM875 SEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS 93TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPAE912 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 94GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAM923 SPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAP 95GSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPAM1318 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS 96TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESXTENAmino Acid Sequence IDNameGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPBC 864 GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGS 97EPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSABD864 GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETA 98SGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSASETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGATSESGAGSETATSGSETAAE948 GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS 99PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPAE1044 SGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT 100STEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEP PCT/U82012/046326{EITEN Amino Acid Sequence IDGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTAE1140 GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGS 101EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAAE1236 GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 102GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAE1332 GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGT 103STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSEXTENAmino Acid Sequence IDNameTPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTAE1428 GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGT 104STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSPAAE1524 GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS 105PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAAE1620 SGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGT 106SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESAGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTAE1716 GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS 107PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSE 2012/046326{EITEN Amino Acid Sequence IDSATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSEAE1812 GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 108STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAE1908 SGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGS 109PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAE2004 GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGS 110A PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP PCT/U82012/046326XTENAmino Acid Sequence IDNameESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSEAG948 GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG 111TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPAG1044 GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG 112TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSSPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTAG1140 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG 113SSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSTAG1236 GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG 114ASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTP PCT/U82012/046326{EITEN Amino Acid Sequence IDSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPAG1332 GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPG 115SSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGAG1428 GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPG 116TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPAG1524 GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG 117TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSXTENAmino Acid Sequence IDNameTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGAG1620 GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPG 118ASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTAG1716 SSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPG 119SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGAG1812 GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG 120SSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPAG1908 GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPG 121 PCT/U82012/046326{EITEN Amino Acid Sequence IDSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPAG2004 STGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG 122A SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPAE72B SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE 123PATSGSETPGAE72C TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS 124TEPSEGSAPGAE108A TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA 125ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSAE108B GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS 126EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPAE144A STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE 127GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSAE144B SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP 128AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGAE180A TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS 129TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSAE2 1 6A TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE 130SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATAE252A ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES 131GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEWO 22617 PCT/U82012/046326XTENAmino Acid Sequence IDNameAEZ88A TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG 132SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAAE324A PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG 133ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSAE360A TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS 134TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATAE396A PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS 135TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSAE432A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE 136SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSAE468A TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE 137SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGTEPSEGSAPGSEPATSGSETPGTSESATAE504A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS 138TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSAE540A TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE 139GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPAE576A TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP 140ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP PCT/U82012/046326XTENAmino Acid Sequence IDNameGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAAE612A GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS 141TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATAE648A PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG 142SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATAE684A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG 143SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSASPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAAE720A PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS 144EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEAE756A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS 145EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPXTENAmino Acid ce IDNameTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAE792A EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE 146SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSAE828A TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE 147SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATAG72A GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS 148PGTPGSGTASSAG72B GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG 149ASSSPAG72C SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSST 150PSGATGSPGAAG108A SASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPG 151TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPAG108B PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP 152GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSAG144A PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 153GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSAG144B PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS 154ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPAG180A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 155TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSAGZ 1 6A SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS 156TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAG252A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 157TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASXTENAmino Acid Sequence IDNameSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGAGZ88A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 158TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSAG324A TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 159STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPAG360A PGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 160STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGAG396A GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT 161ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTAG432A GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG 162ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPAG468A TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 163SSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGAG504A TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 164STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPAG540A TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS 165STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAWO 22617 XTENAmino Acid Sequence IDNameSTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGAG576A TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS 166TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGASTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGAG612A STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT 167GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSAG648A GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG 168ATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPAG684A PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG 169ATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGAG720A TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG 170ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGAG756A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 171TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASXTENAmino Acid Sequence IDNameSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGAG792A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 172ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGAG828A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 173TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPAG288_ GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG 1699DE ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP In other embodiments, the CFXTEN composition ses one or more petitive XTENsequences of lengths ranging from about 36 to about 3000 amino acid residues, wherein at least about80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non-overlapping 36 amino acid sequence motifs selected from one or more of the polypeptide sequences ofTables 13-17, either as a family sequence, or where motifs are selected from two or more families ofmotifs.
In those embodiments wherein the XTEN component of the CFXTEN fusion protein has lessthan 100% of its amino acids consisting of 4, 5, or 6 types of amino acid selected from glycine (G),e (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequenceconsisting of the sequence motifs from Table 3 or the XTEN sequences of Tables 4, and 13-17, the otheramino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but arepreferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. TheXTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) andproline (P) are either interspersed throughout the XTEN sequence, are located within or between thesequence motifs, or are concentrated in one or more short stretches of the XTEN sequence, e. g., to createa linker n the XTEN and the FVIII ents. In such cases where the XTEN component of theCFXTEN comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate(E) and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids behydrophobic es such that the ing sequences generally lack secondary structure, e. g., nothaving more than 2% alpha s or 2% heets, as determined by the methods disclosed herein.
Hydrophobic residues that are less favored in construction ofXTEN include tryptophan, phenylalanine,tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequencesto contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of thefollowing amino acids: cysteine (to avoid disulfide ion and oxidation), methionine (to avoidion), gine and glutamine (to avoid desamidation). Thus, in some embodiments, the XTENcomponent of the CFXTEN fusion protein comprising other amino acids in addition to glycine (G),alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) have a sequence with less than 5% ofthe residues contributing to alpha-helices and beta-sheets as measured by the Chou-Fasman thmand have at least 90%, or at least about 95% or more random coil formation as measured by the GORalgorithm.3. Length of Sequence In r aspect, the invention provides XTEN of varying lengths for incorporation intoCFXTEN compositions wherein the length of the XTEN sequence(s) are chosen based on the property orfunction to be ed in the fusion protein. Depending on the intended property or function, theCFXTEN compositions comprise short or intermediate length XTEN located internal to the FVIHsequence or between FVHI s and/or longer XTEN sequences that can serve as carriers, located inthe fusion proteins as described herein. While not intended to be limiting, the XTEN or fragments ofXTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about3000 amino acid residues. Thus, the XTEN for incorporation into the subject CFXTEN encompassXTEN or fragments ofXTEN with lengths of about 6, or about 12, or about 36, or about 40, or about 42,or about 72 or about 96, or about 144, or about 288, or about 400, or about 500, or about 576, or about600, or about 700, or about 800, or about 864, or about 900, or about 1000, or about 1500, or about 2000,or about 2500, or up to about 3000 amino acid es in length. atively, the XTEN sequencescan be about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400,about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 toabout 3000 amino acid residues in length. The precise length of an XTEN incorporated into the subjectCFXTEN can vary without adversely affecting the activity of a CFXTEN composition. In onement, one or more of the XTEN used in the CFXTEN disclosed herein has 36 amino acids, 42amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and maybe selected from one of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD. Inanother embodiment, two or more of the XTEN used in the CFXTEN disclosed herein has 36 aminoacids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in lengthand may be selected from two of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC orBD, with combinations of AE and AG family sequences preferred. In some embodiments, CFXTENcomprising one or more of the XTEN used herein contain XTEN selected from any one of the sequencesin Table 4, which may be linked to the FVIII component directly or Via spacer ces disclosedherein.
In ular CFXTEN configuration designs, where the XTEN serve as a flexible linker, or areinserted in external loops or unordered regions of the FVIII sequence to increase the bulk, flexibility, orhydrophilicity of the , or are ed to interfere with clearance receptors for FVIII to epharmacokinetic properties, or to interfere with binding of FVIII inhibitors or other VIII antibodies,or where a short or intermediate length ofXTEN is used to facilitate tissue penetration or to vary thestrength of interactions of the CFXTEN fusion protein with its target, or where it is desirable to distributethe cumulative length ofXTEN in segments of short or intermediate length at multiple ons withinthe FVIII sequence, the invention contemplates CFXTEN compositions with one, two, three, four, five ormore short or intermediate XTEN sequences inserted between or within one or more FVIII domains orwithin external loops, or at other sites in the FVIII sequence such as, but not limited to, locations at orproximal to the insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table 9 or asillustrated in FIGS. 8-9. In one embodiment of the foregoing, the CFXTEN fusion protein containsmultiple XTEN segments, e. g., at least two, or at least three, or at least four, or at least five or moreXTEN segments in which the XTEN segments can be identical or they can be different and wherein theCFXTEN retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the procoagulant activity ofnative FVIII when assayed by one of the assays disclosed herein. In other particular CFXTENconfiguration s, where the XTEN serves as a carrier to increase the bulk of the fusion n, or tovary the strength of interactions of the CFXTEN fusion protein with its target, or to enhance thecokinetic ties of the fusion n, the invention contemplates CFXTEN compositionswith one or more intermediate or longer length XTEN sequences inserted at the C-terminus, within the Bdomain (or the residual of the BDD sequence) between or within one or more FVIII domains, withinal loops, or at other sites in the FVIII sequence such as, but not limited to, insertion sites identifiedin Table 5, Table 6, Table 7, Table 8, and Table 9 or as illustrated in FIGS. 8-9. However, it is believedthat the incorporation of multiple XTEN of short to intermediate lengths into CFXTEN compositionsconfers enhanced properties on the fusion proteins compared to CFXTEN fusion proteins with the samenumber of amino acids in fewer but longer length XTEN, yet still results in compositions withprocoagulant activity and extended ife; the ale of which is detailed herein regarding thederived radii of multiple XTEN.
In the ments wherein the CFXTEN fusion proteins comprise multiple XTEN sequences,the tive length of the total residues in the XTEN sequences is greater than about 100 to about3000, or about 200 to about 2000, or about 400 to about 1000 amino acid residues and the XTEN can beidentical or they can be ent in sequence, net charge, or in length. In one embodiment of CFXTENcomprising multiple XTEN, the individual XTEN sequences each exhibit at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% ce identity compared to a motif or an XTEN selected fromTables 3, 4, and 13-17 or a fragment thereof, when lly aligned with a sequence of comparablelength.
As bed more fully below, s are disclosed in which the CFXTEN are designed byselecting the length of the XTEN and its site of oration within the CFXTEN to confer a target half-life, retention of procoagulant actiVity, reduced g to FVIII inhibitors or an enhancedphysicochemical property (e.g., stability or solubility) of a CFXTEN fusion protein, encoding uctsare created and expressed and the recombinant CFXTEN fusion proteins are isolated and recovered. Inl, XTEN cumulative lengths longer that about 400 residues incorporated into the CFXTENitions result in longer half-life compared to shorter cumulative lengths, e.g., shorter than about280 residues. In one embodiment, CFXTEN fusion proteins designs are contemplated that comprise atleast a single XTEN as a carrier, with a long sequence length of at least about 400, or at least about 600,or at least about 800, or at least about 900, or at least about 1000 or more amino acids. In anotherembodiment, multiple XTEN are incorporated into the fusion protein to achieve tive lengths of atleast about 400, or at least about 600, or at least about 800, or at least about 900, or at least about 1000 ormore amino acids, wherein the XTEN can be identical or they can be different in sequence or length. Asused herein, “cumulative length” is intended to encompass the total length, in amino acid residues, whenmore than one XTEN is incorporated into the CFXTEN fusion protein. Both of the ingembodiments are designed to confer increased bioavailability and/or increased terminal half-life afteradministration to a subject compared to CFXTEN comprising shorter cumulative XTEN lengths, yet stillresult in a procoagulant activity and hemostasis effect. When administered subcutaneously orintramuscularly, the Cmax is d but the area under the curve (AUC) is increased in comparison to acomparable dose of a CFXTEN with shorter cumulative length XTEN or FVIII not linked to XTEN,thereby contributing to the ability to maintain effective levels of the CFXTEN composition for a longerperiod of time and permitting increased periods of 2, 4, 7, 10, 14 or 21 days between dosing, asdescribed more fully below. Thus, the XTEN confers the property of a depot to the administeredCFXTEN, in addition to the other physicochemical properties described herein.
When XTEN are used as a carrier, the invention takes advantage of the discovery thatincreasing the length of the non-repetitive, unstructured polypeptides es the unstructured nature ofthe XTENs and correspondingly enhances the physical/chemical and pharmacokinetic properties ofWO 22617 fusion proteins comprising the XTEN carrier. As described more fully in the es, proportionalincreases in the length of the XTEN, even if created by a repeated order of single family ce motifs(e. g., the four AE motifs of Table 3), result in a sequence With a higher percentage (e. g., 90% or more) ofrandom coil formation, as determined by GOR algorithm, or reduced content of alpha-helices or beta-sheets (e. g., less than 2%), as ined by Chou-Fasman algorithm, compared to shorter XTENlengths. In addition, increasing the length of the unstructured polypeptide fusion partner, as described inthe Examples, results in a fusion protein With a disproportionate increase in terminal half-life (e.g., asmuch as 50, 100, 200 or more hours) compared to fusion proteins With unstructured polypeptide partnersWith shorter ce lengths. The enhanced pharmacokinetic properties of the CFXTEN in comparisonto FVIII not linked to XTEN are described more fully, below.
In another aspect, the invention provides methods to create XTEN of short or intermediatelengths from longer ” XTEN sequences, Wherein the longer donor XTEN sequence is ted atthe N—terminus, or the C-terminus, or a fragment is created from the interior of a donor sequence, therebyresulting in a short or intermediate length XTEN. In non-limiting es, as schematically depicted inA—C, an AG sequence of 864 amino acid residues can be truncated to yield an AG sequence With144 residues, an AG sequence With 288 residues, an AG sequence With 576 residues, or otherintermediate lengths, While the AE sequence of 864 residues (as depicted in D, E) can betruncated to yield multiple AE ces of 144 residues, an AE sequence With 288 or 576 residues orother shorter or intermediate lengths. It is specifically contemplated that such an approach can beutilized with any of the XTEN embodiments described herein or with any of the sequences listed inTables 4 or 13-17 to result in XTEN of a desired length. In preferred ments, the CFXTENcomprising multiple XTEN have XTEN exhibiting at least about 80%, or at least about 90%, or at leastabout 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, orat least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequenceidentity to sequences selected from AE42_1, , AE42_3, AG42_1, AG42_2, AG42_3, AG42_4,AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, 4B, AE144_5A,AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, AG144_F, 3, 4,AE288_1, AE288_2, AG288_1, AG288_2, and AG288_DE.4. Net charge In other embodiments, the unstructured characteristic of an XTEN polypeptide can be enhancedby incorporation of amino acid residues with a net charge and/or reduction of the overall percentage (e. g.less than 5%, or 4%, or 3%, or 2%, or 1%) of hydrophobic amino acids in the XTEN sequence. Theoverall net charge and net charge density is lled by modifying the content of charged amino acidsin the XTEN sequences, either positive or negative, With the net charge typically represented as thepercentage of amino acids in the polypeptide contributing to a charged state beyond those residues thatare cancelled by a residue With an opposite charge. In some embodiments, the net charge density of theXTEN of the compositions may be above +0.1 or below -0.1 charges/residue. By “net charge density” ofa protein or peptide herein is meant the net charge diVided by the total number of amino acids in theprotein or propeptide. In other embodiments, the net charge of an XTEN can be about 0%, about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, orabout 20% or more. Based on the net charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the XTEN will have anisoelectric point between 1.5 and 4.5 and carry a net negative charge under physiologic conditions.
Since most tissues and surfaces in a human or animal have a net negative charge, in someembodiments the XTEN sequences are designed to have a net negative charge to ze ecificinteractions between the XTEN ning compositions and various surfaces such as blood vessels,healthy tissues, or various receptors. Not to be bound by a particular theory, an XTEN can adopt openconformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptidethat individually carry a net negative charge and that are distributed across the sequence of the XTENpolypeptide. In some embodiments, the XTEN ce is designed with at least 90% or 95% of thecharged residues separated by other residues such as serine, alanine, threonine, proline or glycine, whichleads to a more uniform bution of charge, better expression or purification behavior. Such adistribution of net negative charge in the extended sequence lengths ofXTEN can lead to an cturedconformation that, in turn, can result in an effective se in hydrodynamic radius. In preferredembodiments, the negative charge of the subject XTEN is conferred by incorporation of glutamic acidresidues. lly, the glutamic residues are spaced uniformly across the XTEN sequence. In somecases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20kDa ofXTEN that can result in an XTEN with charged residues that would have very similar pKa, which canincrease the charge homogeneity of the product and sharpen its isoelectric point, enhance thephysicochemical properties of the resulting CFXTEN filsion protein for, and hence, simplifyingation procedures. For example, where an XTEN with a negative charge is desired, the XTEN canbe selected solely from an AB family sequence, which has approximately a 17% net charge due toincorporated glutamic acid, or can include varying proportions of glutamic acid-containing motifs ofTable 3 to provide the desired degree of net charge. Non-limiting examples of AE XTEN include, butare not limited to the 36, 42, 144, 288, 576, 624, 864, and 912 AB family ces ofTables 4 and 14 orfragments thereof In one embodiment, an XTEN sequence of Tables 4, or 13-17 can be modified toinclude additional glutamic acid residues to e the desired net negative charge. ingly, inone ment the invention provides XTEN in which the XTEN sequences contain about 1%, 2%,4%, 8%, 10%, 15%, 17%, 20%, 25%, or even about 30% glutamic acid. In one ment, theinvention contemplates incorporation of up to 5% aspartic acid residues into XTEN in addition toic acid in order to achieve a net negative charge.
In other embodiments, where no net charge is d, the XTEN can be selected from, forexample, AG XTEN components, such as the AG motifs of Table 3, or those AM motifs of Table 3 thathave no net charge. Non-limiting examples of AG XTEN include, but are not limited to 36, 42, 144,288, 576, and 864 AG family sequences of Tables 4 and 16, or fragments thereof In anotherembodiment, the XTEN can comprise varying proportions of AE and AG motifs (in order to have a netcharge that is deemed l for a given use or to maintain a given physicochemical property.
Not to be bound by a ular , the XTEN of the CFXTEN compositions with thehigher net charge are expected to have less non-specific interactions with various negatively-chargedsurfaces such as blood vessels, tissues, or various receptors, which would further contribute to reducedactive clearance. Conversely, it is ed that the XTEN of the CFXTEN compositions with a low (orno) net charge would have a higher degree of interaction with es that can potentiate the actiVity ofthe associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascularsurfaces to the coagulation process and the ity of activation of coagulation factors (Zhou, R., et al.,Biomaterials (2005) 26(16):2965-2973; , F., et al. Biochemistry (2000) 39(32):9850—9858).
The XTEN of the compositions of the present invention generally have no or a low content ofpositively charged amino acids. In some embodiments, the XTEN may have less than about 10% aminoacid es with a positive , or less than about 7%, or less than about 5%, or less than about 2%,or less than about 1% amino acid residues with a positive charge. However, the invention contemplatesucts where a limited number of amino acids with a positive charge, such as lysine, are incorporatedinto XTEN to permit conjugation between the epsilon amine of the lysine and a reactive group on apeptide, a linker bridge, or a reactive group on a drug or small molecule to be ated to the XTENbackbone. In one embodiment of the ing, the XTEN of the subject CFXTEN has between about 1to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysineresidues, or about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to aboutlysine residues, or about 1 to about 5 lysine residues, or alternatively only a single lysine residue.
Using the foregoing lysine-containing XTEN, fusion proteins can be constructed that comprise XTEN, aFVIII coagulation , plus a chemotherapeutic agent or other coagulation factor or cofactor useful inthe treatment of coagulopathy conditions, wherein the maximum number of molecules of the agentincorporated into the XTEN component is determined by the numbers of lysines or other amino acidswith reactive side chains (e.g., cysteine) incorporated into the XTEN.
As hydrophobic amino acids impart structure to a polypeptide, the invention provides that thecontent of hydrophobic amino acids in the XTEN will typically be less than 5%, or less than 2%, or lessthan 1% hydrophobic amino acid content. In one embodiment, the amino acid content of nine andtryptophan in the XTEN component of a CFXTEN fusion protein is typically less than 5%, or less than2%, and most preferably less than 1%. In another embodiment, the XTEN of the subject CFXTENitions will have a sequence that has less than 10% amino acid residues with a positive charge, orless than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positivecharge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagineand glutamine residues will be less than 5% of the total XTEN sequence.
. Low immunogenicity In another aspect, the XTEN ces provided herein have a low degree of genicityor are substantially non-immunogenic. Several factors can contribute to the low immunogenicity ofWO 22617 XTEN, e. g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, thelow degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence,and the low degree or lack of epitopes in the XTEN sequence.
Conformational es are formed by regions of the protein surface that are composed ofmultiple discontinuous amino acid sequences of the protein antigen. The precise g of the proteinbrings these ces into a well-defined, stable l configurations, or epitopes, that can berecognized as “foreign” by the host humoral immune , resulting in the production of antibodies tothe protein or the activation of a cell-mediated immune response. In the latter case, the immune seto a protein in an individual is heavily influenced by T-cell e recognition that is a function of thepeptide binding specificity of that individual’s HLA—DR allotype. Engagement of a MHC Class IIpeptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-bindingof certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell.tion leads to the release of cytokines further activating other lymphocytes such as B cells toproduce antibodies or activating T killer cells as a full cellular immune response.
The ability of a peptide to bind a given MHC Class II molecule for tation on the surfaceof an APC (antigen presenting cell) is dependent on a number of factors; most notably its primarysequence. In one embodiment, a lower degree of immunogenicity is achieved by designing XTENsequences that resist antigen processing in antigen presenting cells, and/or choosing sequences that donot bind MHC receptors well. The invention provides CFXTEN fusion proteins with ntially non-repetitive XTEN polypeptides designed to reduce binding with MHC II receptors, as well as avoidingformation of epitopes for T-cell receptor or antibody binding, resulting in a low degree ofimmunogenicity. nce of immunogenicity can attribute to, at least in part, a result of theconformational lity ofXTEN sequences; i.e., the lack of secondary structure due to the selectionand order of amino acid es. For example, of ular interest are sequences having a lowtendency to adapt compactly folded conformations in aqueous solution or under physiologic conditionsthat could result in conformational epitopes. The administration of fusion proteins comprising XTEN,using conventional therapeutic practices and dosing, would generally not result in the formation ofneutralizing antibodies to the XTEN sequence, and also reduce the immunogenicity of the FVIII fusionpartner in the CFXTEN compositions.
In one embodiment, the XTEN sequences utilized in the subject fusion proteins can besubstantially free of epitopes recognized by human T cells. The elimination of such epitopes for thepurpose of generating less immunogenic proteins has been disclosed usly; see for e WO98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays forhuman T cell epitopes have been described (Stickler, M., et al. (2003) JImmunol Methods, 281: 95-108).
Of particular interest are peptide sequences that can be oligomerized without generating T cell esor man sequences. This is achieved by g direct repeats of these sequences for the presenceof T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are nothuman, and then altering the design of the XTEN sequence to eliminate or disrupt the e sequence.
WO 22617 In some embodiments, the XTEN sequences are substantially non-immunogenic by the restriction of thenumbers of epitopes of the XTEN predicted to bind MHC receptors. With a reduction in the numbers ofepitopes e of binding to MHC receptors, there is a concomitant reduction in the potential for T cellactivation as well as T cell helper function, reduced B cell activation or upregulation and dantibody production. The low degree of predicted T-cell epitopes can be determined by epitopeprediction algorithms such as, e. g., TEPITOPE (Stumiolo, T., et al. (1999) Nat hnol, 17: 555-61),as shown in Example 46. The TEPITOPE score of a given peptide frame within a protein is the log ofthe Kd (dissociation constant, y, off-rate) of the binding of that peptide frame to multiple of themost common human MHC alleles, as disclosed in Stumiolo, T. et al. (1999) Nature Biotechnology17:555). The score ranges over at least 20 logs, from about 10 to about -10 (corresponding to bindingconstraints of 10e10 Kd to 10e'10 Kd), and can be reduced by avoiding hydrophobic amino acids that serveas anchor residues during peptide display on MHC, such as M, I, L, V, F. In some embodiments, anXTEN ent incorporated into a CFXTEN does not have a predicted T-cell epitope at a PEthreshold score of about -5, or -6, or -7, or -8, or -9, or at a TEPITOPE score of -10. As used herein, ascore of “-9” is a more ent TEPITOPE threshold than a score of -5.
In r embodiment, the inventive XTEN sequences, including those incorporated into thesubject CFXTEN fusion ns, are rendered substantially non-immunogenic by the restriction ofknown proteolytic sites from the sequence of the XTEN, reducing the processing ofXTEN into smallpeptides that can bind to MHC 11 receptors. In another embodiment, the XTEN sequence is renderedntially non-immunogenic by the use a ce that is substantially devoid of secondary structure,conferring resistance to many ses due to the high entropy of the structure. Accordingly, thereduced TEPITOPE score and ation of known proteolytic sites from the XTEN render the XTENcompositions, including the XTEN of the CFXTEN fusion protein compositions, substantially unable tobe bound by mammalian receptors, including those of the immune system or active clearance receptorsthat target FVIII. In one embodiment, an XTEN of a CFXTEN fusion protein can have >100 nM Kdbinding to a mammalian receptor, or greater than 500 nM Kd, or greater than 1 uM Kd towards amammalian cell e receptor or circulating polypeptide receptor.
Additionally, the non-repetitive sequence and corresponding lack of epitopes ofXTEN limitthe ability of B cells to bind to or be activated by XTEN. A repetitive sequence is recognized and canform multivalent contacts with even a few B cells and, as a consequence of the cross-linking of multipleT-cell independent receptors, can stimulate B cell proliferation and antibody tion. In contrast,while an XTEN can make contacts with many different B cells over its extended ce, eachindividual B cell may only make one or a small number of contacts with an individual XTEN due to thelack of repetitiveness of the sequence. Not being to be bound by any theory, XTENs typically have amuch lower tendency to stimulate proliferation of B cells and thus an immune response. In oneembodiment, the CFXTEN have reduced immunogenicity as compared to the corresponding FVIII that isnot fused to an XTEN. In one embodiment, the administration of up to three parenteral doses of aCFXTEN to a mammal result in detectable anti-CFXTEN IgG at a serum dilution of 1:100 but not at adilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of aCFXTEN to a mammal result in detectable anti-FVIII IgG at a serum dilution of 1:100 but not at adilution of 1:1000. In another ment, the administration of up to three parenteral doses of aCFXTEN to a mammal result in able anti-XTEN IgG at a serum dilution of 1:100 but not at adilution of 1:1000. In the foregoing embodiments, the mammal can be a mouse, a rat, a rabbit, or acynomolgus monkey.
An additional feature of XTENs with non-repetitive sequences relative to sequences with ahigh degree of repetitiveness is non-repetitive XTENs form weaker contacts with antibodies. diesare multivalent molecules. For instance, IgGs have two cal binding sites and Ing contain 10identical binding sites. Thus antibodies against repetitive sequences can form multivalent contacts withsuch repetitive sequences with high avidity, which can affect the potency and/or elimination of suchrepetitive sequences. In contrast, antibodies against non-repetitive XTENs may yield monovalentinteractions, resulting in less likelihood of immune clearance such that the CFXTEN compositions canremain in circulation for an increased period of time. In addition, it is believed, as schematicallyportrayed in the flexible unstructured nature of XTEN provides steric shielding of FVIII regionsproximal to the XTEN site of insertion and providing steric hindrance to binding by FVIII inhibitors.
] In another aspect, a subject XTEN useful as a fusion partner has a high hydrodynamic radius; aproperty that in some ments confers a corresponding increased apparent molecular weight to theCFXTEN fusion protein incorporating the XTEN, while in other embodiments enhances steric hindranceto FVIII tors and to anti-FVIII antibodies, reducing their ability to bind to CFXTEN. As detailed inExample 26, the linking ofXTEN to therapeutic n sequences results in CFXTEN compositions thatcan have increased hydrodynamic radii, increased apparent molecular , and increased ntmolecular weight factor compared to a therapeutic protein not linked to an XTEN. For example, intherapeutic applications in which prolonged half-life is desired, compositions in which an XTEN with ahigh hydrodynamic radius is incorporated into a fusion protein comprising a therapeutic protein caneffectively enlarge the hydrodynamic radius of the composition beyond the glomerular pore size ofapproximately 3-5 nm (corresponding to an apparent molecular weight of about 70 kDa) (Caliceti. 2003.
Pharmacokinetic and biodistribution ties of poly(ethylene glycol)-protein ates. Adv DrugDeliv Rev 55 :1261-1277), resulting in reduced renal nce of circulating proteins with acorresponding increase in terminal half-life and other enhanced pharmacokinetic properties. Theynamic radius of a protein is conferred by its molecular weight as well as by its structure,including shape or compactness. Not to be bound by a particular theory, the XTEN can adopt openconformations due to electrostatic repulsion between dual charges of the peptide or the inherentlity imparted by the particular amino acids in the sequence that lack potential to confer secondarystructure. The open, extended and ctured conformation of the XTEN polypeptide can have agreater proportional hydrodynamic radius ed to polypeptides of a comparable ce lengthand/or molecular weight that have secondary and/or ry structure, such as typical globular proteins.
Methods for determining the hydrodynamic radius are well known in the art, such as by the use of sizeWO 22617 2012/046326exclusion chromatography (SEC), as described in US. Patent Nos. 6,406,632 and 7,294,513. Example26 demonstrates that increases in XTEN length result in proportional increase in the hydrodynamicradius, apparent molecular weight, and/or nt molecular weight factor, and thus permit the tailoringof CFXTEN to desired cut-off values of apparent molecular weights or hydrodynamic radii.
Accordingly, in certain embodiments, the CFXTEN fusion n can be configured with an XTEN suchthat the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, orat least about 10 nm, or about 12 nm, or about 15 nm, or about 20 nm, or about 30 nm or more. In theforegoing embodiments, the large hydrodynamic radius conferred by the XTEN in a CFXTEN fusionprotein can lead to reduced clearance of the resulting fusion protein, an increase in terminal half-life, andan increase in mean residence time.
Generally, the actual molecular weight of the mature form of FVIII ent is about 265kDa, while in the case of a FVIII BDD, it is about 165 kDa. The actual molecular weight of a CFXTENfusion protein for comprising a FVIII BDD plus one or more XTEN ranges from about 200 to about 270kDa, depending on the length of the XTEN components. As described in the Examples, when themolecular weights of the CFXTEN fusion proteins are derived from size exclusion chromatographyanalyses, the open conformation of the XTEN due to the low degree of secondary structure results in anincrease in the apparent molecular weight of the fusion proteins into which they are incorporated. Insome embodiments, the CFXTEN comprising a FVIII and at least one or more XTEN exhibits anapparent lar weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, orat least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800kD, orat least about 2000 kD. Accordingly, the CFXTEN fusion proteins comprising one or more XTENexhibit an apparent molecular weight that is about 1.3-fold greater, or about 2-fold greater, or about 3-fold greater or about 4-fold greater, or about 8-fold greater, or about 10-fold greater, or about 12-foldr, or about 15-fold greater than the actual molecular weight of the fusion protein. In oneembodiment, the ed CFXTEN fusion protein of any of the embodiments disclosed herein exhibit anapparent molecular weight factor under logic conditions that is r than about 1.3, or about 2,or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or greater than about 15.
In another embodiment, the CFXTEN fusion protein has, under physiologic conditions, an apparentmolecular weight factor that is about 3 to about 20, or is about 5 to about 15, or is about 8 to about 12, oris about 9 to about 10 relative to the actual molecular weight of the fusion protein. It is believed that theincreased apparent molecular weight of the subject CFXTEN compositions es thepharmacokinetic properties of the fusion proteins by a combination of factors, which e reducedactive clearance, reduced g by FVIII tors, and reduced loss in capillary and venous bleeding.
IV). CFXTEN COMPOSITIONS] The present invention es compositions comprising fusion proteins having factor VIIIlinked to one or more XTEN sequences, wherein the fusion protein acts to replace or augment theamount of existing FVIII in the intrinsic or contact activated coagulation pathway when administeredinto a subject. The invention addresses a elt need in increasing the terminal half-life ofexogenously stered factor VIII to a subject in need f. One way to increase the circulationhalf-life of a therapeutic protein is to ensure that renal clearance or metabolism of the n is reduced.
Another way to increase the terminal half-life is to reduce the active clearance of the therapeutic protein,whether mediated by ors, active metabolism of the protein, or other endogenous mechanisms. Bothmay be achieved by conjugating the protein to a polymer, which, on one hand, is capable of conferring anincreased molecular size (or hydrodynamic radius) to the protein and, hence, reduced renal clearance,and, on the other hand, interferes with g of the protein to clearance receptors or other proteins thatcontribute to metabolism or clearance. Thus, certain objects of the t invention include, but are notlimited to, providing improved FVIII les with a longer circulation or terminal half-life, decreasingthe number or ncy of necessary administrations of FVIII compositions, retaining at least a portionof the ty compared to native ation factor VIII, and/or enhancing the ability to treatcoagulation deficiencies and uncontrolled bleedings more efficiently, more effectively, moreeconomically, and/or with greater safety compared to presently available factor VIII preparations.
Accordingly, the present invention provides recombinant factor VIII fusion proteincompositions comprising an FVIII ntly linked to one or more extended recombinant ptides(“XTEN”), resulting in a CFXTEN fusion protein composition. The term “CFXTEN”, as used herein, ismeant to encompass fusion polypeptides that se at least one payload region comprising a FVIII ora portion of a FVIII that is capable of gulant ty associated with a FVIII coagulation factorand at least one other region comprising one or more XTEN polypeptides that may be interspersed withinthe payload region and/or attached to the terminus. In one embodiment, the FVIII is native FVIII. Inanother embodiment, the FVIII is a sequence variant, fragment, homolog, or mimetic of a naturalsequence that retains at least a portion of the procoagulant actiVity of native FVIII, as disclosed herein.
Non-limiting examples of FVIII suitable for inclusion in the compositions include the sequences of Tablel or sequences having at least 80%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequenceidentity to a sequence of Table 1. In a preferred embodiment, the FVIII is a B-domain deleted (BDD)FVIII sequence variant, such as those BDD sequences from Table l or other such sequences known inthe art. In another preferred embodiment, the CFXTEN comprises a B-domain d (BDD) FVIIIsequence t sed with the native 19 amino acid signal sequence, which is cleaved during thematuration of the protein.
The compositions of the invention include fusion ns that are useful, when administered toa subject in need thereof, for mediating or preventing or ameliorating a condition associated with factorVIII deficiencies or defects in endogenously produced FVIII, or bleeding disorders associated withtrauma, surgery, factor VIII deficiencies or defects. Of particular interest are CFXTEN fusion proteincompositions for which an increase in a pharmacokinetic parameter, increased solubility, increasedstability, or some other ed pharmaceutical property compared to native FVIII is sought, or forwhich increasing the terminal half-life would improve efficacy, safety, or result in reduced dosingfrequency and/or improve patient management. The CFXTEN fusion proteins of the embodimentsdisclosed herein t one or more or any combination of the improved properties and/or theembodiments as detailed herein. In some embodiments, the CFXTEN fusion composition remains at alevel above a threshold value of at least 0.01-0.05, or 0.05 to 0.1, or 0.1 to 0.4 IU/ml when administeredto a subject, for a longer period of time when compared to a FVIII not linked to XTEN and administeredat a comparable dose to a subject in need thereof (e. g., a subject such as a human or mouse or monkeywith hemophilia A).
] The FVIII of the subject compositions, particularly those disclosed in Table 1, together withtheir ponding nucleic acid and amino acid sequences, are available in public databases such asChemical Abstracts Services Databases (e. g., the CAS Registry), GenBank, The sal nResource (UniProt), subscription provided ses such as GenSeq (e.g., Derwent), as well as in thepatent and primary literature. Polynucleotide sequences applicable for expressing the subject CFXTENsequences may be a wild type polynucleotide ce encoding a given FVIH (e. g., either full length ormature), or in some instances the sequence may be a t of the wild type polynucleotide sequence(e. g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNAsequence of the polynucleotide has been optimized, for example, for sion in a particular species, ora polynucleotide ng a variant of the wild type protein, such as a site directed mutant or an allelicvariant. It is well within the ability of the skilled artisan to use a wild-type or consensus cDNA sequenceor a codon-optimized variant of a FVIII to create CFXTEN constructs contemplated by the inventionusing methods known in the art and/or in conjunction with the guidance and methods ed herein,and described more fully in the Examples.
In one embodiment, a CFXTEN fusion protein comprises a single FVIH molecule exhibiting atleast about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to asequence of Table 1 linked to a single XTEN (e. g., an XTEN as described above) including, but notlimited to sequences of the AE or AG family with 42, 144, 288, 576, or 864 amino acids, as set forth inTable 4. In another embodiment, the CFXTEN comprises a single FVIH linked to two XTEN, whereinthe XTEN may be identical or they may be different. In another embodiment, the CFXTEN fusionprotein ses a single FVIH molecule linked to one, two, three, four, five, six or more XTENsequences, in which the FVIII is a sequence that has at least about 80% sequence identity, or alternatively81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1,when optimally d, and the one or more XTEN are each having at least about 80% ceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to one or moresequences selected from any one of Tables 3, 4, and 13-17, when optimally aligned. In the ingembodiment, where the CFXTEN has two or more XTEN, the XTEN may be identical or they may bedifferent sequences. In yet another embodiment, the CFXTEN fusion protein comprises a single FVIH 2012/046326exhibiting at least about 80% ce identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequenceidentity compared to sequences of comparable length selected from Table 1, when optimally aligned,with the portions persed with and linked by three, four, five, six or more XTEN sequences that maybe identical or may be different and wherein each has at least about 80% sequence identity, oralternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to sequences selected fromany one of Tables 3, 4, and 13-17, or nts thereof, when optimally aligned. In yet rembodiment, the invention provides a CFXTEN fusion protein comprising a sequence with at least about80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to a sequencefrom Table 21, when optimally aligned.1. CFXTEN Fusion Protein Configurations The invention provides CFXTEN fusion protein compositions with the CF and XTENcomponents linked in specific N— to inus configurations.
In one embodiment of the CFXTEN composition, the invention provides a fusion protein offormula I:(XTEN)X-CF-(XTEN)y 1wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced fromTable 1; X is either 0 or 1 and y is either 0 or 1 wherein x+y 31; and XTEN is an extended inantpolypeptide as described herein, including, but not limited to sequences having at least about 80%, or atleast about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%,or at least about 99% or 100% sequence identity to sequences set forth in Table 4. Accordingly, theCFXTEN fusion composition can have XTEN-CF, F-XTEN, or CF-XTEN urations.
In another embodiment of the CFXTEN composition, the invention provides a fusion protein offormula II:(XTEN)X-(S)x-(CF)-(XTEN) y 11wherein ndently for each occurrence, CF is a factor VIII as defined herein, including sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forthin Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can allyinclude a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y iseither 0 or 1 wherein x+y 21; and XTEN is an extended inant polypeptide as described hereinincluding, but not limited to sequences having at least about 80%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100%sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion n, wherein the fusion protein is of formula III:(XTEN)X-(S)X-(CF)-(S)y-(XTEN)y 111wherein independently for each ence, CF is a factor VIII as defined herein, including sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequence set for inTable 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionallyinclude a cleavage sequence or amino acids compatible with restrictions sites; X is either 0 or 1 and y iseither 0 or 1 wherein X+y 21; and XTEN is an extended recombinant polypeptide as described hereinincluding, but not limited to sequences having at least about 80%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100%sequence ty to ces set forth in Table 4.
In another embodiment of the CFXTEN composition, the ion provides a recombinantfactor VIII fusion protein of formula IV:(A1)-(XTEN)u-(A2)-(XTEN)V-(B)-(XTEN)W-(A3)-(XTEN)x-(C1)-(XTEN)y-(C2)-(XTEN)Zwherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of theB domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; V is either 0 or 1; w is either 0 or 1;X is either 0 or 1; y is either 0 or 1; y is either 0 or 1 with the proviso that u + V + X + y+z 31; and XTENis an extended recombinant polypeptide as described herein including, but not d to sequenceshaving at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forthin Table 4.
In another embodiment of the CFXTEN composition, the ion provides a recombinantfactor VIII fusion protein of formula V:(XTENx-(sx -(A1)-(S)b-(XTEN)u-(S)b-(A2)-(S)c-(XTEN)V-(S)c-(B)-(S)d-(XTEN)w-(S)d-(A3)—(S)e-(XTEN)x-(S)e-(C1)-(s)r(XTEN)y-(S)r(cz)-(S)g-(XTEN)Z Vwherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice t of theB domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer ce havingbetween 1 to about 50 amino acid residues that can optionally e a cleavage sequence or aminoacids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; V is either 0or 1; w is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t + u + V + w+X + y + z 31; and XTEN is an eXtended recombinant polypeptide as described herein including, but notlimited to sequences haVing at least about 80%, or at least about 90%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identityto sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine ora sequence selected from Tables 11 and 12.
In r embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula VI:(XTENX—(SX-(Al)—(S)b-(XTEN)V-(S)b-(A2)-(S)c-(XTEN)W-(S)c-(A3)—(S)d-(XTEN)x-(S)d-(C1)-(s)e-(XTEN)y-(8)6-(C2)—(S)r(XTEN)Z VIwherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacersequence having between 1 to about 50 amino acid residues that can optionally include a cleavagesequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either0 or 1; dis either 0 or 1; e is either 0 or 1; fis either 0 or 1; uis either 0 or 1; Vis either 0 or 1; Wis 0 or1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u + V + w+ X + y + z 31; andXTEN is an extended recombinant polypeptide as described herein including, but not limited tosequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%,or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity tosequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine or asequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the ion provides a recombinantfactor VIII fusion protein of formula VII:XTEN)X-(CS)x-(S)x-(FVIII_1-745)-(S)y-(XTEN)y-(S)y-(FVIII_1635-2332)-(S)Z-(CS)Z-(XTEN)Z VIIwherein independently for each occurrence, SP is a signal peptide, ably with sequenceMQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is a ge sequence listed in Table 12, S is aspacer sequence having n 1 to about 50 amino acid residues that can optionally include aminoacids compatible with restrictions sites, “FVIII_1-745” is residues 1-745 of Factor FVIII and“FVIII_l635-2332” is residues 1635-2332 of FVIII, X is either 0 or 1, y is either 0 or 1, and z is either 0or 1, wherein x+y+z >2; and XTEN is an eXtended recombinant polypeptide as bed hereinincluding, but not limited to sequences having at least about 80%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100%sequence identity sequences set forth in Table 4. In one embodiment of formula VII, the spacer sequenceis GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine ora ce selected from Tables 11 and 12.
In r embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula VIII:S)a-(XTEN)V-(S)a-(A2)-(B1)—(S)b-(XTEN)w-(S)b-(B2)-(A3)—(S)c-(XTEN)x-(S)c-(C1)—(S)d-y-(S)d-(C2)-(S)e-(XTEN)Z VIIIwherein ndently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII;B1 is a fragment of the B domain that can have from residue 741 to 743-750 of FVIII or alternativelyfrom about residue 741 to about residues 745 of FVIII; B2 is a fragment of the B domain that can havefrom es 1635-1686 to 1689 of FVIII or alternatively from about residue 1640 to about residues1689 of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; Sis a spacer sequence having between 1 to about 50 amino acid residues that can optionally include acleavage sequence or amino acids compatible With restrictions sites; a is either 0 or 1; b is either 0 or 1; cis either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; Wis 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 With the proviso that u + v + w+ X + y + z21; and XTEN is an extended recombinant polypeptide as bed herein including, but not d toces having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%,or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity tosequences set forth in Table 4. In one embodiment of formula VIII, the spacer sequence is GPEGPS(SEQ ID NO: 1612). In another embodiment of a V, the spacer sequence is glycine or a sequenceselected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinantfactor VIII fusion protein of formula IX:(A1N)'(S)a'(XTEN)t'(S)b'(A1C)'(A2 N)-(S)c-(XTEN)u-(S)d-(A2c)-(BN)-(S)e-(XTEN)v-(S)r(Bc)-(A3N)-(S)g'(XTEN)W'(S)h'(A3C)'(C1N)'(S)i'(XTEN)x'(S)j'(C1C)'(C2N)'(S)k'(XTEN)y'(S)1'(C2C)'(S)m'(XTEN)zWherein independently for each occurrence, AlN is a fragment of the A1 domain from at least residuenumber 1 (numbered relative to native, mature FVIII) to no more than residue number 371, A1C is afragment of the A1 domain from at least residue number 2 to no more than residue number 372; A2N is afragment of the A2 domain from at least residue number 373 to no more than residue number 739, Me isa fragment of the A2 domain from at least residue number 374 to no more than residue number 740; BNis a fragment of the B domain from at least residue number 741 to no more than residue number 1647, BCis a fragment of the B domain from at least residue number 742 to no more than residue number 1648;A3N is a fragment of the A3 domain from at least residue number 1649 to no more than residue number2019, A3C is a fragment of the A3 domain from at least e number 1650 to no more than enumber 2019; ClN is a fragment of the C1 domain from at least residue number 2020 to no more thanresidue number 2171, C1C is a fragment of the C1 domain from at least residue number 2021 to no morethan residue number 2172; C2N is a fragment of the C2 domain from at least residue number 2173 to nomore than residue number 2331, C2C is a fragment of the C2 domain from at least residue number 2174to no more than residue number 2332; S is a spacer sequence having between 1 to about 50 amino acides that can optionally include a cleavage sequence or amino acids compatible With restrictionssites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; l is either 0or 1; mis either 0 or 1; t is either 0 or 1; uis either 0 or 1; vis either 0 or 1; Wis 0 or 1, X is either 0 or 1;y is either 0 or 1; z is either 0 or 1 With the proviso that t + u + v + w+ X + y + z 31; and XTEN is aned inant ptide as described herein including, but not limited to sequences having atleast 90% identity to sequences set forth in Table 4. In one embodiment of formula IX, the spacersequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula IX, the spacer sequence isglycine or a sequence selected from Tables 11 and 12.
The embodiments of formulae IV-VIII encompass CFXTEN configurations wherein one ormore XTEN of lengths g from about 6 amino acids to >_1000 amino acids (e. g., sequences selectedfrom any one of Tables 3, 4, and 13-17 or fragments thereof, or sequences exhibiting at least about 90-99% or more sequence identity thereto) are inserted and linked between adjoining domains of the factorVIII or are linked to the N— or C-terminus of the FVIII. In other embodiments of formulae V-VIII, theinvention further provides configurations wherein the XTEN are linked to FVIII domains Via spacersequences which can optionally comprise amino acids compatible with restrictions sites or can includecleavage sequences (e. g., the sequences of Tables 11 and 12, described more fully below) such that theXTEN ng sequence can be, in the case of a restriction site, integrated into a CFXTEN constructand, in the case of a cleavage sequence, the XTEN can be released from the fusion protein by the actionof a protease riate for the ge sequence.
The ments of formulae VI -VIII differ from those of formula V in that the FVIIIcomponent of formulae VI-VIII are only the B-domain deleted forms (“FVIII BDD”) of factor VIII thatretain short residual sequences of the B-domain, non-limiting examples of sequences of which areprovided in Table 1, wherein one or more XTEN or nts ofXTEN of lengths ranging from about 6amino acids to 11000 amino acids (e. g., sequences selected from any one of Tables 3, 4, and 13-17) areinserted and linked between adjoining domains of the factor VIII and/or between the remnants of the Bdomain residues, such as those of Table 8. The ment of formula IX generally differs from thoseof the other formulae in that the one or more XTEN are each inserted within domains of FVIII rather thann domains, and/or has an XTEN linked to the C-terminus of the FVIII (or is linked Via a spacersequence to the C-terminus of the FVIII).
In some embodiments of a CFXTEN, the fusion protein comprises a in deleted form ofFVIII wherein the B-domain deletion starts from a first position at about amino acid residue number 745and ends at a second position at amino acid residue number 1635 to about 1690 with reference to the full-length human factor VIII sequence and an XTEN links the first position and the second position of the B-domain deletion. In one embodiment of the foregoing, the first position and the second position of the B-domain deletion are selected from the positions of Table 8. In another embodiment of the foregoing, atleast one XTEN links the first and second position wherein the at least one XTEN links factor VIII aminoacid e 745 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1640, oramino acid residue 741 and amino acid residue 1690, or amino acid e 745 and amino acid e1667, or amino acid e 745 and amino acid residue 1657, or amino acid residue 745 and amino acidresidue 1657, or amino acid residue 747 and amino acid e 1642, or amino acid residue 751 andamino acid residue 1667. In one embodiment of the , n the factor VIII comprises anXTEN linking a first on and a second position of a B-domain deletion described in theembodiments of this paragraph, the XTEN is a sequence haVing at least 80%, or at least about 90%, or atleast about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%or 100% sequence identity compared to a ce of comparable length selected from any one of Table4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned, wherein the CFXTENretains at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII.
The invention contemplates all possible permutations of insertions ofXTEN between or withinthe domains of FVIII or at or proximal to the insertion points of Table 5, Table 6, Table 7, Table 8, andTable 9 or those illustrated in FIGS. 8-9, with optional linking of an additional XTEN to the N— or C-terminus of the FVIII, optionally linked via an additional cleavage sequence selected from Table 12,resulting in a CFXTEN ition; miting examples of which are portrayed in FIGS. 5 and 12.
In one ment, the CFXTEN comprises a FVIII BDD sequence of Table 1 in which one or moreXTEN that each has at least about 80%, or at least about 90%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99% or more sequence identitycompared to a sequence from any one of Tables 3, 4, and 13-17 or fragments thereof are insertedbetween any two of the residual B domain amino acids of the FVIII BDD sequence, resulting in a singlechain FVIII fusion protein, wherein the CFXTEN retains at least about 30%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%of the procoagulant activity of native FVIII. In the ing embodiment, the CFXTEN can have anadditional XTEN sequence of any one of Tables 4, and 13-17 linked to the N— or C-terminus of the fusionprotein. In another embodiment, a CFXTEN comprises at least a first XTEN inserted at a site set forth inTable 8, wherein the CFXTEN retains at least about 30%, or at least about 40%, or at least about 50%, orat least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulantactivity of native FVIII. In one embodiment of a fusion protein of formula VII, the CFXTEN comprisesa FVIII BDD sequence of Table 1 in which two or more XTEN that each has at least about 80%, or atleast about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99%, or 100% ce identity compared to a sequence from any one of Tables3, 4, and 13-17 or nts thereof are linked to a FVIII-BDD sequence in which at least one XTEN isinserted from about 3 to about 20 amino acid residues to the C-terminus side of the FVIII cleavage siteamino acid R740 and from about 3 to about 20 amino acid es to the N—terminus side of the FVIIIcleavage site amino acid R1689 of the residual B domain amino acids of the FVIII BDD sequence,resulting in a single chain FVIII fusion protein, and one or two XTEN are linked by a cleavage sequenceto the N— and/or C-terminus of the FVIII-BDD sequence, wherein the CFXTEN ts at least about40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at leastabout 90% of the procoagulant activity of native FVIII after release of the XTEN by cleavage of thecleavage sequences.
In one ment, the A3 domain comprises an a3 acidic region or a portion thereof. Inanother ment, at least one XTEN is inserted within the a3 acidic region or the portion thereof, N-terminus of the a3 acidic region or the n thereof, C-terminus of the a3 acidic region or the portionWO 22617 thereof, or a combination thereof. In certain embodiments, at least one XTEN is inserted within the C2domain, N—terminus of C2 domain, C-terminus of C2 domain, or a ation thereof. In still otherembodiments, the Factor VIII comprises all or portion of B domain. In yet other embodiments, at leastone XTEN is inserted within all or a portion of B domain, N—terminus of B domain, C-terminus of Bdomain, or a combination thereof2. CFXTEN Fusion Protein Configurations with Internal XTEN In r aspect, the invention provides CFXTEN configured with one or more XTENsequences located internal to the FVIII ce. In one embodiment, invention provides CFXTENconfigured with one or more XTEN sequences located internal to the FVIII ce to confer propertiessuch as, but not limited to, increased stability, increased resistance to proteases, increased resistance toclearance mechanisms including but not limiting to interaction with clearance ors or FVIIIinhibitors, and increased hydrophilicity, compared to FVIII without the incorporated XTEN.
The invention contemplates that different configurations or sequence variants of FVIII can beutilized as the platform into which one or more XTEN are ed. These configurations include, but arenot limited to, native FVIII, FVIII BDD, and single chain FVIII (scFVIII), and variants of thoseconfigurations. In the case of scFVIII, the invention provides CFXTEN that can be constructed byreplacing one or multiple amino acids of the processing site of FVIII. In one embodiment, the scFVIIIed in the CFXTEN is created by replacing the R1648 in the FVIII sequence TR (SEQ IDNO: 1698) with glycine or alanine to prevent proteolytic sing to the heterodimer form. It isspecifically contemplated that any of the CFXTEN embodiments disclosed herein with a 1648 FVIIIresidue can have a glycine or alanine substitution for the arginine at position 1648. In someembodiments, the invention provides CFXTEN comprising scFVIII wherein parts of the sequencesurrounding the R1648 processing site are replaced with XTEN, as illustrated in FIGS. 10A and 10B. Inone embodiment, at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97% or more of the B-domain is ed with an XTEN sequence disclosed herein, including one ormore of the R740, R1648, or R1689 cleavage sites. In another embodiment, the CFXTEN has the FVIIIsequence of the B-domain between the FXIa ge sites at R740 and R1689 (with at least 1-5 adjacentB-domain amino acids also retained between the cut site and the start of the XTEN to permit the seto access the cut site) ed with XTEN. In another embodiment, the CFXTEN has the FVIIIce of the B-domain between the FXIa cleavage site at N745 and P1640 replaced with XTEN. Inother embodiments, the ion provides CFXTEN FVIII BDD sequence variants in which portions ofthe B-domain are d but only one of the FXI R740 or R1689 activation sites (and 1-5 adjacentamino acids of the B-domain) are left within the construct, wherein the XTEN remains attached at oneend to either the light or heavy chain after cleavage by FXIa, as illustrated in and 5D. In oneembodiment of the foregoing, the CFXTEN comprises a FVIII BDD ce in which the amino acidsbetween N745 to P1640 or between S743 to Q1638 or between P747 to V1642 or between N745 andQ1656 or between N745 and S1657 or between N745 and T1667 or between N745 and Q1686 orbetween R747 and V1642 or between T751 and T1667 are deleted and an XTEN sequence is linkedbetween these amino acids, connecting the heavy and light chains, and can further se additionalXTEN inserted either in external surface loops, between FVIII domains, or at the N— or C-termini of theFVIII BDD sequence, such as one or more insertion sites from Table 5, Table 6, Table 7, Table 8, andTable 9 or those illustrated in FIGS. 8-9. In another embodiment of the foregoing, the CFXTENcomprises a FVIII BDD sequence in which the amino acids between K713 to Q1686 or between residues741 and 1648 are deleted and an XTEN linked between the two amino acids, and additional XTEN canbe inserted either in e loops, between FVIII domains, or at the N— or ini of the FVIII BDDsequence, including but not limited to one or more insertion sites from Table 5, Table 6, Table 7, Table 8,and Table 9 or those illustrated in FIGS. 8-9. In some embodiments such CFXTEN ces can haveone or more XTEN exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at leastabout 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identityto an XTEN sequence from any one of Tables 4 and 13-17.
] The invention contemplates other CFXTEN with al XTEN in s urations;schematics of exemplary configurations are illustrated in FIGS. 5 and 10. The regions suitable for XTENinsertion sites include the known domain ries of FVIII, exon boundaries, known surface (external)loops and solvent accessible surface area sites identified by X-ray crystallography analysis, and structuremodels derived from molecular c tions of FVIII, s with a low degree of order(assessed by programs described in FIGS. 7 legend), regions of low homology/lack of conservationacross different species, and hydrophilic regions. In another embodiment, XTEN insertion sites wereselected based on FVIII putative clearance receptor binding sites. In another embodiment, CFXTENcomprises XTEN inserted at ons not within close proximity to mutations implicated in hemophiliaA listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database wereeliminated (Kemball-Cook G, et al. The factor VIII Structure and Mutation Resource Site: HAMSTeRSversion 4. Nucleic Acids Res. (1998) 26(1):216-219). In another embodiment, potential sites for XTENinsertion include residues within FVIII epitopes that are capable of being bound by anti-FVIII antibodiesoccurring in sensitized hemophiliacs and that do not otherwise serve as protein interactive sites. Regionsand/or sites that are considered for exclusion as XTEN insertion sites include es/regions of factorVIII that are important in various ctions ing other clotting proteins, residues surrounding eacharginine activating/inactivating cleavage site acted on by the proteases thrombin, factor Xa, activatedprotein C, residues surrounding the signal peptide processing site (residue 1) if the construct contains thesignal peptide, regions known to interact with other proteins such as FIXa, FX/FXa, in, activatedprotein C, protein S cofactor to Protein C, von Willebrand , sites known to interact withphospholipid cofactors in coagulation, residues involved in domain interactions, residues coordinatingCa++ or Cu++ ions, cysteine residues involved in S-S intramolecular bonds, nted amino acidinsertion and point mutation sites in FVIII ed in hemophilia A subjects affecting procoagulantactivity, and mutation sites in FVIII made in a research lab that affect procoagulant activity. Sitesconsidered for either insertion (to prolong half-life) or for exclusion (needed to remove spent FVIIIa orFXa) e regions known to interact with n sulfate proteoglycan (HSPG) or low-densitylip oprotein receptor-related protein (LPR).
By analysis of the foregoing criteria, as described in Example 34, different insertion sites orranges of insertions sites across the FVIII BDD sequence have been identified and/or confirmed asates for insertion of XTEN, non-limiting examples of which are listed in Table 5, Table 6, Table 7,Table 8, and Table 9 and are shown schematically in FIGS. 8 and 9. In one embodiment, CFXTENcomprise XTEN insertions between the individual domains of FVIII, i.e., between the Al and A2, orn the A2 and the B, or between the B and the A3, or between the A3 and the C1, or n theCl and the C2 s. In r embodiment, CFXTEN comprises XTEN inserted within the Bdomain or between remnant residues of the BDD sequence. In another embodiment, CFXTEN comprisesXTEN inserted at known exon boundaries of the encoding FVIII gene as exons represent evolutionaryconserved sequence modules that have a high probability of functioning in the context of other proteinsequences. In another embodiment, CFXTEN comprise XTEN inserted within surface loops identifiedby the x-ray structure of FVIII. In another embodiment, CFXTEN comprise XTEN inserted withinregions of low order identified as having low or no detected electron density by X-ray structure is.
In another embodiment, CFXTEN se XTEN inserted within regions of low order, predicted bystructure prediction algorithms such as, but not limited to dex, RONN, and Kyte & llethms. In another embodiment, CFXTEN comprise XTEN inserted within sequence areas of highfrequency of hydrophilic amino acids. In another embodiment, CFXTEN comprise XTEN inserted withinepitopes e of being bound by naturally-occurring anti-FVIII antibodies in sensitized hemophiliacs.
In another embodiment, CFXTEN comprise XTEN inserted within sequence areas of low ceconservation and/or differences in sequence segment length across FVIII sequences from differentspecies. In another embodiment, CFXTEN comprise XTEN linked to the N—terminus and/or C-terminus.
In r embodiment, the invention provides CFXTEN configurations with inserted XTEN selectedfrom two or more of the ia from the embodiments listed above. In another embodiment, theinvention provides CFXTEN configurations with at least one, atively at least two, alternatively atleast three, alternatively at least four, alternatively at least five or more XTEN inserted into a factor VIIIsequence wherein the points of insertion are at or proximal to the N— or C-terminus side of the at leastone, two, three, four, or five, or six or more amino acids selected from the insertion residue amino acidsof Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9, or alternatively withinone, or within two, or within three, or within four, or within five, or within six amino acids of theinsertion residue amino acids from Table 5, Table 6, Table 7, Table 8, and Table 9, or within the variousspans of the insertion residue amino acids schematically portrayed for an exemplary FVIII BDDsequence in As described above, the one or more intemally—located XTEN or a fragment ofXTEN can havea sequence length of 6 to 1000 or more amino acid residues. In some embodiments, wherein theCFXTEN have one or two or three or four or five or more XTEN sequences internal to the FVIII, theXTEN sequences can be identical or can be different. In one embodiment, each internally-locatedXTEN has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identitycompared to comparable lengths or fragments ofXTEN or motifs selected from any one of Tables 3, 4,and 13-17, when optimally aligned. In another embodiment, the invention provides a CFXTENconfigured with one or more XTEN inserted internal to a FVIII BDD sequence with at least about 80%sequence identity, or atively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1,wherein the insertions are located at the insertion points or range of insertion points indicated in Table 5,Table 6, Table 7, Table 8, and Table 9, or within the range of insertions as illustrated in Itwill be understood by those of skill in the art that an XTEN ed within the FVIII sequence at aninsertion point of Table 5, Table 6, Table 7, Table 8, and Table 9 is linked by its N— and C-termini toflanking FVIH amino acids (or via a linking spacer or cleavage ces, as bed above), while anXTEN linked to the N— or C-terminus of FVIII would only be linked to a single FVIII amino acid (or to alinking spacer or cleavage sequence amino acid, as described above). By way of example only,variations of CFXTEN with three internal XTEN could have: XTEN (as described herein) incorporatedn FVHI BDD residues 741 and 1640, residues 18 and 19, and es 1656 and 1657; or XTENincorporated between FVHI BDD residues 741 and 1640, residues 1900 and 1901, and at the C-terminusat residue 2332; or XTEN incorporated between FVHI BDD residues 26 and 27, residues 1656 and 1657,and residues 1900 and 1901; or XTEN incorporated n FVHI BDD residues 741 and 1640, residues1900 and 1901, and at the C-terminus at residue 2332.
In evaluating the CFXTEN fusion ns with XTEN inserted in the locations from Table 5, itwas discovered that insertions in certain regions of the FVIII sequence resulted in CFXTEN with goodexpression and retention of procoagulant activity. Accordingly, in preferred embodiments, the ionprovides CFXTEN fusion ns configured with one, or two, or three, or four, or five, or six or moreXTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequencety compared to an XTEN selected from any one of Tables 4, and 13-17 inserted internal or linked toa FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%ce identity compared to a sequence of Table 1, wherein the insertions are located at an insertionpoint within one, or two, or three, or four, or five, or six or more ranges set forth in Table 7. 1n theforegoing embodiments, the CFXTEN fusion ns with the XTEN insertions retain at least about%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the procoagulant activity compared tothe corresponding FVIII not linked to XTEN.
In evaluating the CFXTEN fusion ns with XTEN inserted in one or more locations fromTable 5, it was surprisingly discovered that a high tage of fusion proteins with the XTENinsertions retained procoagulant activity, as described in Example 25. Accordingly, the inventionprovides CFXTEN fusion proteins configured with one, two, three, four, five, six or more XTEN whereinWO 22617 the resulting fusion protein exhibits at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or70%, or 80%, or 90% or more of the procoagulant activity compared to the corresponding FVIII notlinked to XTEN when assayed by a coagulation assay described herein. In a preferred embodiment, theinvention provides CFXTEN fusion proteins sing one, or two, or three, or four, or five, or six ormore XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity compared to an XTEN selected from any one of Tables 4, and 13-17 linked to a FVIIIBDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequencety compared to a sequence of Table 1, wherein the insertions are located at one or more insertionpoints selected from Table 5, Table 6, Table 7, Table 8, and Table 9, and wherein the resulting fusionprotein exhibits at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, orat least about 70% or more procoagulant activity compared to the corresponding FVIII not linked toXTEN, when assayed in vitro by an assay bed herein (e. g., a chromogenic assay). As the subjectCFXTEN fusion proteins typically exhibit increased terminal half-life compared to native FVHI, it willbe appreciated by one of skill in the art that a CFXTEN with lower procoagulant activity relative to anequimolar amount of native FVIH would nevertheless be acceptable when administered as a therapeuticition to a subject in need therof. In another embodiment, the CFXTEN fusion proteinssing one, or two, or three, or four, or five or more XTEN, each having at least about 80%sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an XTEN selected fromany one of Tables 4, and 13-17 linked to a FVIII BDD sequence with at least about 80% sequenceidentity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein theinsertions are d at one or more ion points or the range of insertion points selected from Table, Table 6, Table 7, Table 8, and Table 9, wherein the resulting fusion n exhibits at least about 0.5IU/ml, or at least about 0.75 lU/ml, or at least about 1.0 lU/ml, or at least about 1.5 lU/ml, or at leastabout 2.0 IU/ml, or at least about 2.5 lU/ml, or at least about 3 lU/ml, or at least about 4 lU/ml, or at leastabout 5 lU/ml, or at least about 7 lU/ml, or at least about 10 lU/ml, or at least about 20 lU/ml, or at leastabout 30 lU/ml FVIII activity when expressed in cell culture medium and assayed in a chromogenicassay, wherein the culture and expression are according to methods described herein; e.g., the methods ofe 25.
It is believed that the discovery of the insertions sites wherein the FVIH s at least aportion of its procoagulant activity would also permit the insertion of other peptides and polypeptideswith either unstructured or structured characteristics that are ated with the prolongation of half-lifewhen fused to a FVIII protein in one or more of those same sites. Non-limiting examples includealbumin, n fragments, Fc fragments of immunoglobulins, the B subunit of the C-terminal peptide(CTP) of human chorionic gonadotropin, a HAP sequence, a transferrin, the PAS polypeptides of US.
Pat Application No. 20100292130, polyglycine linkers, polyserine linkers, peptides and shortpolypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A),serine (S), threonine (T), glutamate (E) and proline (P) with varying degrees of secondary structure fromless than 50% to greater than 50%, amongst , would be suitable for insertion in the identified activeinsertions sites of FVIII.
In the fusion protein embodiments described herein, the CFXTEN fusion protein can fithhercomprise one or more cleavage sequence from Table 12 or other sequences known in the art, thecleavage sequence being located between or within 6 amino acid es of the intersection of the FVIIIand the XTEN ces, which may include two cleavage sequences in a given internal XTENsequence. In one embodiment, the CFXTEN comprising cleavage ces has two identical cleavagesequences, each located at or near the respective ends of one or more internal XTEN such that the XTENis released from the fusion protein when cleaved by the protease that binds to and cleaves that sequence.
The sequences that can be cleaved are described more fully below and exemplary sequences are providedin Table 12.
Table 5: Insertion ons for XTEN linked to the FVIII BDD seguenceFVIH BDDXTEN Insertion Insertion FVH?N0. . . Downstream DomalnP01nt ResumeSequence1 0 (N-terminus) ATR A12 3 R RYY A13 17 M QSD A14 18 Q SDL A122 G ELP A16 24 L PVD A17 26 V DAR A18 28 A RFP A19 32 P RVP A13 8 F PFN A111 40 F NTS A112 41 N TSV A113 60 N IAK A114 61 I AKP A165 R PPW A116 81 Y DTV A117 111 G AEY A118 116 D QTS A119 119 S QRE A1120 Q REK A121 128 V FPG A122 129 F PGG A123 130 P GGS A124 182 G SLA A1185 A KEK A126 188 K TQT A127 205 G KSW A128 210 S ETK A129 211 E TKN A1216 L MQD A131 220 R DAA A132 222 A ASA A1W0 22617 FVHI BDDXTEN Insertion Insertion FVH?Pomt. ReSIdue.
Downstream Domaln—Seb33 223 A SAR A134 224 s ARA A1230 K MHT A136 243 p GL1 A137 244 G L1G A13 8 250 R KSV A139 318 D GME A140 333 P QLR A142 334 Q LRM A143 336 R MKN a144 339 N NEE a145 345 D YDD a146 357 V VRF a147 367 S FIQ a148 370 s RPY a149 375 A KKH A250 376 K KHP A251 378 H PKT A252 399 V LAP A253 403 D DRS A254 405 R SYK A255 409 S QYL A256 416 P QRI A257 434 E TFK A258 43 8 T REA A259 441 A IQH A260 442 1 QHE A261 463 I IFK A262 487 Y SRR A263 490 R LPK A264 492 P KGV A265 493 K GVK A266 494 G VKH A267 500 D FPI A268 506 G EIF A269 518 E DGP A270 556 K ESV A271 565 Q IMS A272 566 I MSD A273 598 P AGV A274 599 A GVQ A275 603 L EDP A276 616 s ING A277 686 G LWI A278 713 K NTG A279 719 Y EDS A280 730 L LSK A281 733 K NNA A282 745 N PPV B83 1640 P PVL B84 1652 R TTL B85 1656 Q SDQ A386 1685 N QSP A3FVHI BDDXTEN Insertion Insertion FVH?P01nt. ReSIdue.
Downstream Domaln87 171 1 M SSS A388 1713 S SPH A389 1720 N RAQ A390 1724 S GSV A391 1725 G SVP A392 1726 S VPQ A393 1741 G SFT A394 1744 T QPL A395 1749 R GEL A396 1773 V TFR A397 1792 Y EED A398 1793 E EDQ A399 1796 Q RQG A3100 1798 Q GAE A3101 1799 G AEP A3102 1802 P RKN A3103 1803 R KNF A3104 1807 V KPN A3105 1808 K PNE A3106 1827 K DEF A3107 1844 E KDV A3108 1861 N TLN A3109 1863 L NPA A31 10 1896 E RNC A31 1 1 1900 R APC A31 12 1904 N IQM A31 13 1905 I QME A31 14 1910 P TFK A31 15 1920 A ING A31 16 1937 D QRI A31 17 1981 G VFE A31 18 2019 N KCQ A31 19 2020 K CQT C1120 2044 G QWA C1121 2068 F SW1 C1122 2073 V DLL C1123 2090 R QKF C1124 2092 K FSS C1125 2093 F SSL C1126 21 1 1 K WQT C1127 21 15 Y RGN C1128 2120 T GTL C1129 2125 V FFG C1130 2171 L NSC C1131 2173 S CSM C2132 2188 A QIT C2133 2223 V NNP C2134 2224 N NPK C2135 2227 K EWL C2136 2268 G HQW C2137 2277 N GKV C213 8 2278 G KVK C2139 2290 F TPV C2140 2332 Y C terminus of FVIII CTIndicates an insertion point for XTEN based on the amino acid number of mature full-length humanFVHI, wherein the insertion could be either on the N- or C-terminal side of the indicated amino acidDownstream sequence in FVIII BDD with 746-1639 deletionTable 6. Exemplagy insertion locations for XTEN linked to a FVIII polypeptideFVHI BDDXTEN Insertion FVIII D1§t3“°6.fr°mNo. . . Downstream insertionInsertion Point . Domain.
Se .uence residue9 32 P RVP A1 -3, --631 220 R DAA A1 -34 224 S ARA A1 +543 336 R MKN a1 -1, --644 339 N NEE a1 -4, --552 399 V LAP A2 -6, ——356 416 P QRI A2 +675 603 L EDP A2 _6, --685 1656 Q SDQ B —3, --687 1711 M SSS A3 -6, --191 1725 G SVP A3 --61 13 1905 I QME A3 --6114 1910 P TFK A3 -5, --6Distance from insertion residue refers to the ve number of amino acids away from the N-terminus(negative numbers) or C-terminus (positive numbers) of the designated insertion residue (residue “0”)where an insertion may be made. The designation “-X” refers to an insertion site which is X amino acidsaway on the inal side of the designated insertion residue. Similarly, the designation “+X” refersto an insertion site which is X amino acids away on the C-terminal side of the designated insertionFor e, “-1, +2” indicates that the insertion is made at the N-terminus or C-terminus of aminoacid residues denoted -1, 0, +1 or +2.
Table 7. Further exemplagy insertion locations for XTEN linked to a FVIII polypeptideXTEN Insertion First ion FVIII DomainPoint Range Residue3 18-32 Q A18 40 F A118 21 1-224 E A127 336-403 R A1, A243 599 A A247 745-1640 N B50 1656-1728 Q B, A357 1796-1804 R A365 1900-1912 R A381 2171-2332 L C1,C2indicates range of insertion sites ed relative to the amino acid number of mature human FVIII PCT/U82012/046326Table 8. Exemplary XTEN insertion locations within B-domain deleted variants of a FVIIIpolypeptideXTEN Insertion First Insertion Second InsertionPoint Range Residue Residue740-1640 R P740-1690 R S741-1648 S R743-163 8 S Q745-163 8 N Q745-1640 N P745-1656 N Q745-1657 N S745-1667 N T86 N Q42 R V751-1667 T Tindicates the amino acids linked Within the B-domain deleted variant and nt A3 domain, Withthe amino acids numbered relative to the amino acid number of mature human FVIIIindicates the amino acids linked by an XTEN inserted in the BDD-FVHITable 9. Exemplary insertion locations for XTEN linked to a FVIII polypeptide resulting inprocoagulant activityA F 111 BDD F IIIXTEN InsertlonN0. ion ReSidue Dywnmam DanainPoint——_____%gm_—2 3 R RYY A14 18 Q SDL A122 G ELP A17 26 V DAR A111 40 F NTS A118 116 D QTS A119 119 S QRE A126 188 K TQT A129 211 E TKN A1216 L MQD A131 220 R DAA A134 224 S ARA A1230 K MHT A140 333 P QLR A143 336 R MKN a144 339 N NEE a152 399 V LAP A253 403 D DRS A255 409 s QYL A256 416 P QRI A260 442 l QHE A262 487 Y SRR A263 490 R LPK A266 494 G VKH A269 518 E DGP A274 599 A GVQ A275 603 L EDP A2 2012/046326. FVIII BDD FVIIIXTEN InsertionNo. Insertion e ream DomainPomtSequence78 713 K NTG A282 745 N PPV B85 1656 Q SDQ A387 1711 M SSS A389 1720 N RAQ A391 1725 G SVP A399 1796 Q RQG A3102 1802 P RKN A3110 1896 E RNC A3111 1900 R APC A3112 1904 N IQM A3113 1905 I QME A3114 1910 P TFK A3121 2068 F SWI C1130 2171 L NSC C1135 2227 K EWL C2137 2277 N GKV C2140 2332 Y C terminus of FVIII C2Downstream ce in FVIII BDD with 39 deletion In another aspect, the invention provides libraries of components and methods to create thelibraries d from nucleotides encoding FVIII segments, XTEN, and FVIII segments linked to XTENthat are useful in the preparation of genes encoding the subject CFXTEN. In a first step, a library ofgenes encoding FVIII and XTEN inserted into the various single sites at or within 1-6 amino acids of aninsertion site identified in Table 5 or illustrated in FIGS. 8-9 are created, expressed, and the CFXTENrecovered and evaluated for activity and pharmacokinetics as illustrated in . Those CFXTENshowing enhanced properties are then used to create genes encoding a FVIII segment and the insertionsite plus an XTEN, with components from each ed insertion represented in the library, asillustrated in . In one embodiment, the library components are assembled using standardrecombinant techniques in combinatorial n, as rated in , resulting in permutations ofCFXTEN with multiple internal and N- and C-terminus XTEN, that can include the insertion sites of orproximal to those Table 5, Table 6, Table 7, Table 8 and Table 9, or as illustrated in FIGS. 8-9. Theresulting constructs would then be evaluated for activity and enhanced pharmacokinetics, and thosecandidates resulting in CFXTEN with enhanced properties, e.g., d active clearance, resistance toproteases, reduced immunogenicity, and enhance pharmacokinetics, compared to FVIII not linked toXTEN, are evaluated further.3. XTEN Permissive Loops As described in detail elsewhere herein and as illustrated in FIGS.33-3 6, the inventors haverecognized that each FVIII “A” domain comprise at least two “XTEN permissive loops” into whichXTEN sequences can be inserted without eliminating procoagulant activity of the recombinant protein, orthe ability of the recombinant proteins to be expressed in Vivo or in Vitro in a host cell. The inventorshave identified the XTEN permissive loops as regions with, among other attributes, high surface orWO 22617 solvent exposure and high conformational flexibility. The Al domain comprises an XTEN permissiveloop-l (Al -1) region and an XTEN permissive loop-2 (Al -2) region, the A2 domain comprises an XTENpermissive loop-l (A2-l) region and an XTEN sive loop-2 (A2-2) , the A3 domaincomprises an XTEN permissive loop-l (A3-l) region and an XTEN permissive loop-2 (A3-2) region..
In certain aspects a recombinant FVIII protein as described above comprises at least one XTENsequence inserted into at least one of the XTEN permissive loops Al-l, A1-2, A2-1, A2-2, A3-1, or A3-2, wherein the recombinant FVIII protein has procoagulant ty and can be expressed in Vivo or inVitro in a host cell. In n aspects a recombinant FVIII protein as described above comprises at leasttwo XTEN sequences inserted into FVIII, e. g., into two different XTEN permissive loops Al-l, A1-2,A2-l, A2-2, A3-l, or A3 -2, wherein the recombinant FVIII protein has procoagulant activity and can besed in Vivo or in Vitro in a host cell. Alternatively, a recombinant FVIII protein as described abovecan comprise two or more XTEN sequences inserted into a single XTEN permissive loop either with ourwithout XTEN sequences inserted into other XTEN permissive loops, n the recombinant FVIIIprotein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a host cell. In certain aspectsa recombinant FVIII protein as bed above can comprise at least one XTEN sequence ed intoat least one of the XTEN permissive loops as described above, and can further comprise one or moreXTEN sequences inserted into a3, wherein the recombinant FVIII protein has procoagulant actiVity andcan be expressed in Vivo or in Vitro in a host cell. In certain aspects, a recombinant FVIII protein of theinvention can comprise three, four, five, six or more XTEN sequences inserted into one or more XTENpermissive loops or into a3, wherein the recombinant FVIII protein has procoagulant actiVity and can beexpressed in Vivo or in Vitro in a host cell.
In certain aspects a recombinant FVIII protein as bed above ses at least one XTENsequence inserted into a3, wherein the recombinant FVIII protein has procoagulant actiVity and can beexpressed in Vivo or in Vitro in a host cell. In certain aspects a recombinant FVIII protein of theinvention comprises at least one XTEN sequence inserted into a3, and further comprises one or moreXTEN ces inserted into one or more XTEN permissive loops as described above, wherein therecombinant FVIII protein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a hostcell.
The inventors have recognized that a recombinant FVIII protein of the invention comprises atleast two XTEN permissive loops in each of the FVIII A domain regions which allows for insertion of anXTEN sequence while haVing procoagulant actiVity and still being able to be expressed in Vivo or in Vitroby a host cell. Various crystal structures of FVIII have been determined, of varying degrees ofresolution. These structures of FVIII and FVIIIa, determined by X-ray crystallography and molecularc simulation, were used to generate models of accessible e area and mationalflexibility for FVIII. For example, the crystal structure of human FVIII has been determined by Shen etal. Blood 111: 1240-1247 (2008) and Ngo et al. Structure 16: 597-606 (2008). The data for thesestructures is available from the Protein Data Bank (pdb.org) under Accession s 2R7E and 3CDZ,respectively.
The predicted secondary structure of the heavy and light chains of human FVIII according tothe Shen et al. crystal structure is reproduced in FIGS. 37A and 37B. The various beta strands predictedfrom the Shen et al. crystal structure are numbered consecutively in FIGS. 8A and 8B. In certainembodiments, the XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, and A3 -2 are contained withinsurface-exposed, flexible loop structures in the A domains of FVIII. A1-1 is located between beta strand1 and beta strand 2, A1-2 is located between beta strand 11 and beta strand 12, A2-1 is located betweenbeta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-1 isd between beta strand 38 and beta strand 39 and A3-2 is located n beta strand 45 and betastrand 46, according to the ary ure of mature FVIII stored as Accession Number 2R7E of thePDB database (PDB:2R7E) and as shown in FIGS. 8A and 8B. The secondary structure of PDBAccession Number 2R7E shown in FIGS. 8A and 8B corresponds to the standardized ary structureassignment ing to the DSSP program (Kabsch and Sander, Biopolymers, 22:2577-2637 (1983)).
The DSSP secondary structure of the mature FVIII stored as PDB Accession Number 2R7E can beaccessed at the DSSP database, available at the world wide web site swift.cmbi.ru.n1/gV/dssp/ (lastaccessed February 9, 2012) (Joosten et al., 39(Suppl. 1): D411-D419 (2010)).
In n aspects, a surface-exposed, flexible loop structure comprising A1-1 corresponds to aregion in native mature human FVIII from about amino acid 15 to about amino acid 45 of . Incertain aspects, A1-1 corresponds to a region in native mature human FVIII from about amino acid 18 toabout amino acid 41 of . In certain aspects, the surface-exposed, flexible loop structuresing A1-2 corresponds to a region in native mature human FVIII from about amino acid 201 toabout amino acid 232 of . In certain aspects, A1-2 corresponds to a region in native maturehuman FVIII from about amino acid 218 to about amino acid 229 of . In certain aspects, thesurface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature humanFVIII from about amino acid 395 to about amino acid 421 of . In certain aspects, A2-1corresponds to a region in native mature human FVIII from about amino acid 397 to about amino acid418 of . In certain aspects, the surface-exposed, flexible loop structure comprising A2-2ponds to a region in native mature human FVIII from about amino acid 577 to about amino acid635 of . In certain aspects, A2-2 corresponds to a region in native mature human FVIII fromabout amino acid 595 to about amino acid 607 of . In certain aspects, the surface-exposed,flexible loop ure sing A3-1 corresponds to a region in native mature human FVIII fromabout amino acid 1705 to about amino acid 1732 of . In n aspects, A3-1 corresponds to aregion in native mature human FVIII from about amino acid 1711 to about amino acid 1725 of .
In certain s, the surface-exposed, flexible loop structure comprising A3-2 ponds to a regionin native mature human FVIII from about amino acid 1884 to about amino acid 1917 of Incertain aspects, A3 -2 corresponds to a region in native mature human FVIII from about amino acid 1899to about amino acid 1911 of .
In certain aspects a recombinant FVIII protein of the invention comprises one or more XTENsequences inserted into one or more XTEN permissive loops of FVIII, or into the a3 region, wherein therecombinant FVIII protein has procoagulant activity and can be sed in Vivo or in Vitro in a hostcell. XTEN sequences to be inserted include those that increase the in Vivo half-life or the in Vivo or inVitro stability of FVIII.
In certain aspects, a recombinant FVIII protein of the invention comprises an XTEN sequencesinserted immediately downstream of one or more amino acids corresponding to one or more amino acidsin mature native human FVIII including, but not limited to: amino acid 18 of , amino acid 26 of, amino acid 40 of , amino acid 220 of , amino acid 224 of , amino acid399 of , amino acid 403 of , amino acid 599 of , amino acid 603 of , aminoacid 1711 of , amino acid 1720 of , amino acid 1725 of , amino acid 1900 of , amino acid 1905 of , amino acid 1910 of , or any ation thereof, includingcorresponding insertions in EDD-variants of FVIII described herein.
In certain aspects, a recombinant FVIII protein of the invention comprises at least one XTENsequence inserted into the a3 region of FVIII, either alone or in ation with one or more XTENsequences being inserted into the XTEN permissive loops of the A domains (e. g., Al-l, A1-2, A2-1, A2-2, A3-1, or A3 -2 as described above), wherein the inant FVIII protein has procoagulant actiVityand can be expressed in Vivo or in Vitro in a host cell. In certain aspects, at least one XTEN sequence isinserted into the a3 region immediately downstream of an amino acid which corresponds to amino acid1656 of . In certain aspects, a recombinant FVIII protein of the invention ses an XTENsequence ed into the a3 region as described, and further includes one or more XTEN sequencesinserted immediately downstream of one or more amino acids corresponding to one or more amino acidsin mature native human FVIII including, but not limited to: amino acid 18 of , amino acid 26 of, amino acid 40 of , amino acid 220 of , amino acid 224 of , amino acid399 of , amino acid 403 of , amino acid 599 of , amino acid 603 of , aminoacid 1711 of , amino acid 1720 of , amino acid 1725 of , amino acid 1900 of , amino acid 1905 of , amino acid 1910 of , or any combination thereof.
It will be understood by one of skill in the art that the foregoing s of permissive loops ofa native FVIII protein into which a heterologous n can be inserted are also applicable to the B-domain deleted FVIII variants described herein; e. g., sequences set forth in Table 1. In practicing thepresent ion, it will be understood that a BDD-FVIII sequence of Table 1 can be substituted for therecombinant FVIII n of the various embodiments described above, and it is ed that theresulting constructs will similarly retain procoagulant actiVity.4. Interference with FVIII binding agents It is an object of the present invention to provide procoagulant CFXTEN fusion proteincompositions for use in human patients suffering from coagulopathies, such as haemophilia A, who havenative or acquired antibodies, tors, or other proteins or molecules that bind to FVIII that affect theactiVity or half-life of CFXTEN fusion proteins, n the CFXTEN retain a greater amount ofprocoagulant ty compared to the corresponding FVIII not linked to XTEN. As used herein, “FVIIIbinding agent” means any molecule capable of binding to native FVIII or to a recombinant factor VIIIfusion protein of the invention comprising factor VIII or a fragment thereof, whether native, derived, orproduced inantly. It is specifically contemplated that FVIII binding agent includes anti-FVIIIantibodies and FVIII inhibitors, amongst other proteins capable of specifically binding to FVIII. In oneaspect, the invention provides procoagulant CFXTEN fiJsion proteins that exhibit d binding to ananti-FVIII antibody or FVIII inhibitor that eres with the procoagulant activity of FVIII. As usedherein, “anti-FVIII antibody” or “anti-factor VIII antibody” means an antibody capable of binding FVIIIor a FVIII component of a CFXTEN of the invention, said dy including but not limited to theantibodies of Table 10 or polyclonal antibody from a hemophilia A t with FVIII inhibitors. Theterm antibody es monoclonal antibodies, polyclonal antibodies, antibody fragments and antibodyfragment clones. As used herein, “FVIII inhibitor” or “anti-FVIII inhibitor antibody” means an antibodycapable of binding FVIII or a FVIII component of a CFXTEN of the invention and that reduces by anymeans the procoagulant actiVity of FVIII or the FVIII component of a CFXTEN. In another aspect, theinvention provides CFXTEN fusion ns that retain procoagulant actiVity in the presence of a FVIIIinhibitor. In another aspect, the invention provides CFXTEN fusion proteins comprising FVIII thatexhibit increased terminal half-life in the presence of a FVIII g agent ed to the FVIII notlinked to XTEN.
] The majority of inhibitory antibodies to human factor VIII act by binding to epitopes located inthe A2 domain or the C2 domain of factor VIII, disrupting specific functions associated with theses, (US. Patent No. 6,770,744; Fulcher et al. Localization of human factor FVIII inhibitor epitopesto two ptide fragments. Proc. Natl. Acad. Sci. USA (1985) 82:7728-7732; Scandella et al. Epitopemapping of human factor VIII inhibitor antibodies by deletion analysis of fVIII fragments expressed inEscherichia coli. Proc. Natl. Acad. Sci. USA (1988) 85:6152-6156). While 68% percent of inhibitorydies are reported to be directed against the A2 and/or C2 domain, 3% act against the A1 domainand 46% against the a3 acidic region (LaVigne-Lissalde, G., et al. Characteristics, mechanisms of ,and epitope mapping of actor VIII antibodies. Clin ReV Allergy Immunol (2009) 37:67-79). Forexample, certain heavy chain-specific inhibitors react with the 18.3-kD amino-terminal segment of theA2 domain (Scandella D, et al. 198 8); Lollar P et al. Inhibition of human factor VIIIa by anti-A2 subunitantibodies. J Clin Invest 1994;93:2497). FVIII contains a phospholipid binding site in the C2 domainn amino acids 2302 and 2332, and there is also a von Willebrand factor binding site in the C2domain that acts in conjunction with amino acids 1649-1689 in the A3 . The C2 domain also hasepitopes that, when bound by inhibitors, block the activation of FVIII by thrombin or factor Xa.
Inhibitors g specifically to the light chain recognize epitopes in the A3 domain or a major antigenicregion in the C2 domain and can result in reduced procoagulant actiVity by preventing the binding ofFVIII to phospholipid or reducing the dissociation rate of FVIII from von Willebrand factor (Gilles JG, etal. Anti-factor VIII dies of hemophiliac patients are frequently directed s nonfunctionaldeterminants and do not exhibit isotypic restriction. Blood (1993) 82:2452; Shima M, et al. A factor VIIIneutralizing monoclonal antibody and a human tor alloantibody recognizing epitopes in the C2domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost(1993) 69:240). Non-limiting examples of monoclonal FVIII tors are listed in Table 9. In patientswith high-titer tors, there is an increased risk of developing recurrent bleeding in particular joints,which may ultimately result in decreased quality of life, disability, or death from excessive blood loss(US. Pat. Application No. 20120065077; Zhang et al., Clinic. Rev. Allerg. Immunol., 37:114-124; Gouw and van den Berg, Semin. Thromb. Hemost., 35 :723-734 (2009)) While not intending to be bound by any ular theory, it is believed that the cturedcharacteristic of the XTEN incorporated into the CFXTEN fusion proteins permits the XTEN to adoptconformations that result in steric hindrance to inhibitors that would otherwise bind to FVIII epitopes.
As illustrated in as the incorporated XTEN assumes various random coil conformations, itspatially covers regions of the FVIII component of the fusion n and sterically interferes with theability of an inhibitor to bind to a FVIII epitope.
In one embodiment, the invention provides CFXTEN exhibiting procoagulant ty andreduced binding in the presence of an antibody binding to the C2 domain of factor VIII compared to thecorresponding factor VIII not linked to XTEN and/or to native FVIII. In another embodiment, theinvention provides CFXTEN ting procoagulant activity and reduced binding in the presence of andy g to the A2 domain of Factor VIII compared to the corresponding factor VIII not linkedto XTEN or to native FVIII. In another embodiment, the invention provides CFXTEN exhibitingprocoagulant activity and reduced binding in the presence of antibodies binding to the A2 and the C2domain of Factor VIII, compared to the corresponding factor VIII not linked to XTEN or to native FVIII.
In one embodiment, the invention provides CFXTEN exhibiting procoagulant activity and reducedg, compared to the ponding FVIII not linked to XTEN, in the presence of an antibodyselected from the group consisting of the antibodies of Table 10. In one embodiment, the CFXTENfusion protein exhibits reduced binding to the dy GMA8021. In another embodiment, theCFXTEN fusion protein exhibits d binding to the antibody GMA8008. In another embodiment,the CFXTEN fusion protein exhibits reduced binding to the antibody ESH4. In another embodiment, theCFXTEN fusion protein exhibits reduced binding to the antibody ESH8. In another embodiment, theCFXTEN fusion protein exhibits d binding to the antibody B02C11. In another ment, theCFXTEN fusion protein exhibits reduced binding and a greater degree of procoagulant activity,ed to the corresponding FVIII not linked to XTEN, in the presence of plasma from a hemophiliaA t with polyclonal antibody FVIII inhibitors, wherein the r degree of procoagulant activityis determined by an in Vitro assay such as a Bethesda assay or other assay described herein.
The CFXTEN exhibiting reduced binding by FVIII inhibitors can have one, or two, or three, orfour, or five, or six or more individual XTEN, embodiments of which are disclosed herein. In theforegoing embodiments of this paragraph, a CFXTEN exhibits at least 5%, or 10%, or 15%, or 20%, or%, or 40%, or 50%, or 60%, or 70% or less binding to the antibody when assessed in Vitro in an assaycapable of assaying the binding of an antibody to FVIII, such as assays described herein below or thoseknown in the art. Alternatively, the reduced binding of the subject CFXTEN to the bindingantibodies can be assessed by retention of a higher degree of gulant activity in the presence of the 2012/046326antibody compared to FVIII not linked to XTEN, as described in the Examples. Thus, in theembodiments ning to reduced binding by FVIII tors described herein, a CFXTEN ts,when reacted with the anti-FVIH antibody, at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or50%, or 60%, or 70%, or 80%, or 100%, or 200%, or 300%, or 400%, or 500% or more activity in aation assay (such as described herein below) compared to the corresponding FVIII not linked toXTEN and reacted with the antibody. In the foregoing, the anti-FVHI antibody can be an antibody fromTable 9 or a circulating anti-FVHI antibody from a hemophilia A subject. In another embodiment, theinvention provides CFXTEN in which the assayed fusion protein, when assayed utilizing the Bethesdaassay and an anti-FVHI antibody selected from Table 10 or a polyclonal anti-FVHI antibody ationsuch as, but not limited to, plasma from a hemophilia A subject with FVIII inhibitors, results in aBethesda titer with at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 fewerBethesda units compared to a FVIII not linked to XTEN and assayed under comparable conditions. Inanother embodiment, the invention provides CFXTEN in which the assayed fusion protein results in lessthan 50%, or less than 40%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, orless than 14%, or less than 13%, or less than 12%, or less than 11%, or less than 10% of the BethesdaUnits ed to a FVIII not linked to XTEN when assayed under comparable conditions utilizing theda assay and a polyclonal anti-FVHI antibody preparation such as, but not limited to, plasma froma hemophilia A subject with FVHI inhibitors.
Table 10: Anti-factor VIII antibodiesAntibody Epitope Inhibitor Titer ReferenceDesignation BU/mgC2 Domain US. 6,770,744BOZCH 20000Met2199/Phe2200 Blood (2007) 110:4234—4242C2 DomainNMC VIII-5 US. 6,770,744Glu2181-Val2243ESH2 Light Chain ADILight Chain US. 744ESH4 392303-2332 Blood (2007) 110:4234—4242C2 Domain US. 6,770,744ESH8 100002248-2285 Blood {2007} 110:4234—4242RHDS(LMBP C1 DomainUS Pat. Appllcatlon 200902633806165CB;. US Pat. ation 20090263380LE2E9 C1 DomamBlood (2000) 95:156-163154 C2 Domain 1300 Blood (2007) 110:4234—4242F85 C2 Domain 6 Blood (2007) 110:4234—4242131 .00 C2 Domain 5 Blood 2007 110:4234—4242E137 C2 Domain 6 Blood (2007) 34—4242189 C2 Domain 1900 Blood (2007) 110:4234—42421117 C2 Domain 1800 Blood (2007) 110:4234—4242ll 09 Meg9137;111:23200 1500 Blood (2007) 110:4234—42421135 C2 Domain 930 Blood (2007) 110:4234—42423C6 C2 Domain 71 Blood (2007) 110:4234—424231312 (3131:5113? 2600 Blood (2007) 110:4234—4242Antibody Epitope Inhibitor Titer ReferenceDesignation BU/mg13102 C2 Domain 3800 Blood 2007 110:4234—42423G6 C2 Domain 25000 Blood (2007) 110:4234—42422—77 C2 Domain 25000 Blood (2007) 110:4234—4242B45 C2 Domain 21000 Blood (2007) 110:4234—4242B9 C2 Domain 31000 Blood 2007 110:4234—42421311 C2 Domain 3300 Blood 2007 110:4234—4242B75 C2 Domain Indeterminate Blood (2007) 110:4234—42423105 Valg2213712nzl2n227 08 Blood (2007) 110:4234—4242F77 C2 Domain 26000 Blood 2007 34—424213178 C2 Domain 18000 Blood (2007) 110:4234—4242F67 C2 Domain 21000 Blood (2007) 110:4234—4242(399 13712nzl2n227 15000 Blood (2007) 110:4234—4242(386 C2 Domain 4300 Blood (2007) 34—4242114 C2 Domain 44000 Blood (2007) 110:4234—4242155 C2 Domain 10000 Blood (2007) 110:4234—42422»—--l 17 C2 Domain >0.4 Blood 2007 34—4242A2 domainGMAOIZ GVIA497-510; 584-593GVIA8001 A3 Domain 1 5 6 GVIAGVIA8002 A1 Domain <1 GVIAGVIA8003 C2 Domain GVIAGVIA8004 A1 Domain GVIAGVIA8005 A1A3/A1 Domain GVIAGVIA8006 c2 Domain GVIA08 C2 Domain 1047 GVIAGVIA8009 A2 Domain 7923 GVIAGVIA801 0 LC Domain GVIAGVIA801 1 C1 Domain 97 GVIAGVIA8012 A1A3 Domain 204 GVIAGVIA8013 A3C2 Domain 3 0 GVIAGVIA8014 C2 Domain 7799 GVIAGVIA8015 A2 Domain 17079 GVIAGVIA801 6 A2 Domain <1 GVIA17 A2 Domain 3 34 GVIAGVIA801 8 LC Domain 242 GVIAGVIA8019 CR-LC Domain GVIAGVIA8020 A1A3 Domain 196 GVIAGVIA8021 A2 Domain 33928 GVIA4A4 A2 Domain 40000 231mm) Haemost (2009) 7.658-3136 C2 Domain 41 Blood (2007) 110:4234—4242an Diagnostica Inc. internet site, URL located on the World Wide Web atamericandiagnostica.c0m/html/Pr0duct_Detail.asp?idCategorF5&idSubCategor3F104&idpr0=ESH-8 asit existed on January 12, 2012Green Mountain Antibodies internet site, URL located on the World Wide Web atgreenmoab.com/product_details/16316/215 82.html as it existed on January 12, 2012 Assays For Inhibitor and Antibody Binding The fusion ns of the invention may be assayed to confirm reduced binding by FVIIIinhibitors using methods known in the art. The assays that can be used include, but are not d to,competitive and non-competitive assay systems using techniques such as Western blots,radioimmunoassays, ELISA, “sandwich” immunoassays, precipitation assays, precipitinreactions, gel diffusion itin reactions, immunodiffusion assays, agglutination assays,immunoradiometric assays, fluorescent immunoassays, clotting assays, factor VIII tor assays toname but a few. Such assays are routine and well known in the art (see, e. g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which isorated by reference herein in its entirety). Exemplary are described briefly below but are notintended by way of limitation.
The Bethesda assay and the Nijmegen modification of the Bethesda assay are factor VIIIinhibitor assays well-known as methods to detect FVIII inhibitors (Kasper CK, et al. Proceedings: Amore uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. (1975) 34(2):612).
However, the assays can be modified to assay binding of inhibitors to FVIII compositions usinginhibitors such as onal or monoclonal anti-FVIII antibodies, including the antibodies of Table 10,and methods such as described in e 52. Briefly, the modified Bethesda assay involves mixingtitered volumes of the test sample with an equal volume of an inhibitor at a set concentration. Themixtures are incubated for 2 hours at 37°C prior to analysis of the factor concentration by a coagulationassay such as a chromogenic assay. rly, a reference plasma with native factor VIII level isted that then assayed as the ve control. The nt is the titer resulting in 50% of the FVIIIactivity of the positive control, ed as Bethesda units. In the Nijimegen modification of theBethesda assay, the assay samples are stabilized with imidazole buffer and the control sample is mixedwith deficient plasma instead of buffer (Verbruggen B, et al. The en modification of the Bethesdaassay for factor VIII:C inhibitors: ed city and reliability. Thromb Haemost. (1995)73(2):247-251).
Western blot analysis generally comprises preparing protein samples, electrophoresis of theprotein samples in a rylamide gel (e. g., 8%—20% GE depending on the molecular weightof the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such asnitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e. g., PBS with 3% BSA ornon-fat milk), washing the membrane in washing buffer (e. g., PBS-Tween 20), blocking the newith primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane inwashing buffer, blocking the membrane with a secondary antibody (which recognizes the primaryantibody, e. g., an anti-human antibody) conjugated to an enzymatic substrate (e. g., horseradishperoxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blockingbuffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill inthe art would be dgeable as to the parameters that can be modified to increase the signal detectedand to reduce the background noise. For further discussion regarding western blot protocols see, e. g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NewYork at 10.8.1.
ELISA assays can detect antibodies to FVIII independent of their ability to block theprocoagulant activity of FVIII, and have been ed for the ion of VIII ping inhemophilia A patients. In a population of 131 ts with hemophilia A with inhibitors, the ELISAtechnique resulted in 97.7% ivity and 78.8% specificity, and had a high negative predictive value(98.6%) [Martin, P. G., et al. Evaluation of a novel ELISA screening test for detection of factor VIIIinhibitory antibodies in haemophiliacs. Clin Lab ol (1999) 21 :125-128]. Other investigators havefound a highly significant correlation between the Bethesda titer and the absorbance values in an ELISAassay for detecting anti-FVIII Abs (Towfighi, F., et al. Comparative measurement of anti-factor VIIIantibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope city. ActaHaematol (2005) 114:84-90), with the added advantage of the ability to detect non-inhibitory VIIIantibodies. Assay protocols comprise preparing the binding ligand, which may include a samplecomprising either factor VIII polypeptide or the CFXTEN fusion protein, g the well of a 96 wellmicrotiter plate with the antibody, adding the ligand test sample and incubating, then adding a detectionantibody and incubating prior to washing and adding a alkaline atase- or peroxidase-conjugatedary antibody and incubating for an additional period before the addition of TMB substrate andprocessing for reading by spectrophotometer at 450nm. In ELISAs the antibody or inhibitor of interestdoes not have to be conjugated to a detectable compound; instead, a second antibody (which recognizesthe antibody or inhibitor of interest) conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antibody, the ligand may be coated to the well. One of skillin the art would be knowledgeable as to the parameters that can be modified to increase the signaldetected as well as other variations of ELISAs known in the art (see, e. g., Ausubel et al, eds, 1994,Current Protocols in lar Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).
Standard or modified coagulation assays are used to measure d binding of FVIII bindingagents. In one exemplary method (further described in Example 28), the optimal concentration of agiven FVIII inhibitor to utilize in the assay is first determined by a titration experiment using varyingamounts of the inhibitory antibody incubated at 37°C for 2 hrs with the base vector expressing ypeFVIII containing a His/Myc double tag. The FVIII ty is measured by the Coatest assay uredescribed herein. The lowest concentration that results in optimal inhibition of FVIII activity isemployed in the assay. In the assay, the FVIII inhibitor antibody at the optimal concentration is mixedwith individual test samples and incubated at 37°C for 2 hrs. The resulting test s are thented and utilized in the Coatest activity assay, along with untreated aliquots of the CFXTEN andpositive control in order to assess the al and baseline FVIII activity for each test sample.
The invention provides methods of making CFXTEN that exhibit reduced binding to FVIIIbinding agents, including FVIII inhibitors, and retention of gulant activity. In one embodiment,the method to make a CFXTEN with reduced binding to FVIII inhibitors comprises the steps of selectinga FVIII sequence with at least 90% sequence identity to a sequence of Table 1, selecting one, two, three,four, five, or six or more XTEN each with at least 70%, or at least 80%, or at least 90%, or at least 95 -99% sequence identity to XTEN sequences of comparable length from Table 4, creating expressionconstructs designed to locate said XTEN at or proximal to locations selected from Table 5, Table 6,Table 7, Table 8, and Table 9, expressing and recovering the ing CFXTEN, and assaying theresulting fusion ns in an assay bed herein in order to confirm the reduced binding of theCFXTEN fusion protein. By the inventive method, a CFXTEN exhibits at least 5% reduced, or at least% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40%reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80%reduced g to a FVIII binding agent including, but not limited to the antibodies of Table 10 or anti-FVIII antibodies from a hemophilia A subj ect, and retains at least about 10%, or at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%procoagulant actiVity compared to the ponding FVIII not linked to XTEN.
Up to 8-10% of hemophilia A patients have antibodies that bind FVIII without affecting itsprocoagulant properties; they are not, therefore rized as FVIII inhibitors. However, the binding ofdies to FVIII is believed to lead to immune complexes that are cleared by the innate immuneresponse or are more susceptible to proteolytic degradation (Kazatchkine MD. Circulating immunecomplexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A. Clin ExpImmunol. (1980) 39(2):315-320). Accordingly, it is an object of the invention to provide CFXTENfusion proteins comprising one or more XTEN that t reduced g of antibodies to FVIII thatare not inhibitors, wherein the ation or clearance of the CFXTEN is reduced at least 5%, or 10%,or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or less compared to a corresponding FVIII notlinked to XTEN or to native FVIII bound by such antibodies. The reduced binding of dies toCFXTEN compared to FVIII not linked to XTEN or to native FVIII can be assayed by in Vitro and invivo methods. In Vitro s include the aforementioned ELISA and Western blot methods. Thereduced degradation or clearance of CFXTEN can be assessed in vivo by use of animal models or inhuman clinical trials. In one type of trial, factor VIII or CFXTEN are administered separately,preferably by intravenous infusion, to cohorts of patients haVing factor VIII deficiency who haveantibodies that promote degradation or clearance of eutic human factor VIII. The dosage of theadministered test article is in a range between 5 and 50 IU/kg body weight, ably 10-45 IU/kg, andmost preferably 40 IU/kg body weight. Approximately 1 hour after each administration, the recovery offactor VIII or CFXTEN from blood samples is measured in a functional one-stage or chromogeniccoagulation assay to assess actiVity and by ELISA, HPLC, or similar assay to qualify the amount of intactfactor VIII equivalent. Samples are taken again approximately 5-10 hours after infusion, and recovery ismeasured. Total recovery and the rate of disappearance of factor VIII from the samples is predictive ofthe antibody titer, and the comparison of results from the factor VIII and CFXTEN indicates the degreeof reduced clearance and/or degradation of the CFXTEN. In one embodiment, the CFXTEN fusionn exhibits at least 5% reduced, or at least 10% d, or at least 15% reduced, or at least 20%reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60%reduced, or at least 70% reduced, or at least 80% reduced binding to an anti-FVIII antibody that promotesclearance but does not otherwise inhibit the procoagulant activity of intact native FVIII. In anotherembodiment, the CFXTEN fusion protein exhibits at least 5% reduced, or at least 10% reduced, or atleast 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least50% d, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to ananti-FVIII antibody that promotes the degradation of FVIII. In the foregoing embodiments of thisparagraph, the reduced binding of the anti-FVIII antibody is alternatively characterized by an increasedKD value of the FVIII antibody to the fusion protein compared to the FVIII of at least ld, or three-fold, or old, or five-fold, or 10-fold, or 33-fold, or 100-fold, or 330-fold, or at least 1000-folded to the g to the corresponding FVIII not linked to XTEN. In one embodiment, theCFXTEN fusion proteins comprising one or more XTEN exhibiting reduced reactivity to an anti-FVIIIantibody exhibits an increased terminal ife when administered to a subject with anti-FVIIIantibodies of at least 48 h, or at least 72 h, or at least 96 h, or at least 120 h, or at least 144 h, or at least14 days, or at least 21 days compared to FVIII not linked to XTEN. In the ing ment, thesubject can be a human hemophilia A subject or it can be a mouse hemophilia A subject with circulatinganti-FVIII antibodies.
Another aspect of the present invention is the use of CFXTEN fusion protein for a specifictherapy of a coagulopathy in a subject with a FVIII inhibitor. The invention provides a method oftreating a subject with circulating FVIII tor(s) sing the step of administering a clotting-effective amount of a CFXTEN fusion protein to the subject wherein the fusion protein exhibits rgulant ty and/or clotting-effective concentrations of longer duration compared to either acorresponding factor VIII not linked to XTEN or compared to native factor VIII administered to thesubject using a comparable amount and route of administration. In one embodiment of the method, theFVIII inhibitor in the subject is an anti-FVIII antibody. In another embodiment, the FVIII inhibitor is aneutralizing anti-FVIII antibody. In one embodiment, the FVIII tor is an VIII antibody thatbinds to the A1 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibodythat binds to the A2 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIIIantibody that binds to the A3 domain of FVIII. In r embodiment, the FVIII tor is an anti-FVIII antibody that binds to the C1 domain of FVIII. In another embodiment, the FVIII inhibitor is ananti-FVIII antibody that binds to the C2 domain of FVIII. In r embodiment, the FVIII inhibitor isan anti-FVIII antibody that binds to both the C2 and A2 domain of FVIII. In another embodiment, theFVIII inhibitor binds to a FVIII epitope capable of being bound by one or more antibodies of Table 10.
In another embodiment, the FVIII inhibitor is a polyclonal antibody from a hemophilia A subject withFVIII inhibitor antibodies.
An obj ect of the present invention is the creation of CFXTEN with XTEN inserted to maximizethe steric interference of FVIII binding agents that would otherwise bind to FVIII and neutralizeprocoagulant activity or result in the clearance or degradation of FVIII. Accordingly, in one approachthe invention provides CFXTEN comprising one or more XTEN wherein the XTEN are insertedproximal to a binding site of a FVIII inhibitor or anti-FVIII antibody. In one embodiment, an XTEN islinked to the FVIII at a on selected from Table 5, Table 6, Table 7, Table 8, and Table 9 that iswithin about 50, or about 100, or about 150, or about 200, or about 250, or about 300 amino acids of aFVIII epitope that is bound by an antibody of Table 10. In another embodiment, the XTEN is linked tothe FVIII within about 50, or about 100, or about 150, or about 200, or about 250, or about 300 aminoacids of a FVIII epitope in the A2 or C2 domain that is bound by an antibody of Table 10. Accordingly,the ion provides CFXTEN fusion proteins comprising one or more XTEN wherein binding byFVIII inhibitors to the FVIII component of the fusion n is reduced compared to the correspondingFVIII not linked to XTEN or to native FVIII and the CFXTEN retains procoagulant actiVity. In theforegoing embodiments hereinabove described in this paragraph, the fusion proteins can be assayed bythe assays described herein below, the assays of the Examples, or other assays known in the art, and theinhibitors can be an dy of Table 10, can be polyclonal anti-FVIII, or can be blood or plasma from ahemophilia A subject with FVIII inhibitors.
In r aspect, CFXTEN are designed to maximize the regions over which XTEN can adoptrandom coil conformations covering the fusion protein, thereby resulting in steric hindrance for anti-FVIII antibodies that would otherwise bind epitopes on the FVIII component of the fusion protein. It isbelieved that the incorporation of multiple XTEN into a CFXTEN provides a higher total hydrodynamicradius of the XTEN component compared to CFXTEN with fewer XTEN yet haVing approximately thesame total ofXTEN amino acids. Empirically, the hydrodynamic radius for a protein can be calculatedbased on size exclusion chromatography, and results of several fusion proteins using such methods aredescribed in the Examples. Alternatively, the radius for XTEN polypeptides, such as those incorporatedin the embodiments disclosed herein, can be approximated by mathematical formulae because the limitedtypes of amino acids utilized have known teristics that can be quantified. In one embodiment, them radius of a single XTEN polypeptide is calculated (hereinafter “XTEN Radius”) according tothe formula given by Equation II:XTEN Radius = N length 0.2037) + 3.4627 11 In another embodiment, the sum of the m of the XTEN Radii for all XTEN segments ina CFXTEN is calculated (hereinafter “Sum XTEN Radii”) according to the formula given by EquationIII:Z XTEN i=1Sum XTEN Radii = 111wherein: n = the number ofXTEN segmentsand i is an iterator In another embodiment, the ratio of the SUM XTEN Radii of a CFXTEN comprising multipleXTEN to that of an XTEN Radius for a single XTEN of an lent length (in total amino acides to that of the ) is calculated (hereinafter “Ratio XTEN Radii”) according to the formulagiven by Equation IV:W0 2013/122617 [L1 XTEN Radiusi.1 [L1XTEN Length] * 0.2037) + 3.4627 Ratio XTEN Radii = ( IVwherein: n = the number ofXTEN segmentsand i is an iterator] In applying the Equations to the XTEN, it will be understood by one of skill in the art that thecalculated values represent maximum values that could vary or be reduced depending on the host cellutilized for expression of the XTEN polypeptide. It is believed that while E. coli expression would resultin XTEN that achieves the calculated values, expression in eukaryotic host cells in which XTEN may beglycosylated could result in a radius of the polypeptide less than the maximum calculated value. Suchdifferences can be quantified by methods such as size exclusion chromatography, the s of whichare detailed in the Examples.
In order to design CFTEN that maximize the area over which XTEN can adopt random coilconformations, it was ered that CFXTEN s with Ratio XTEN Radii above 2 provide greatercoverage over the fusion protein than designs with values <2. Accordingly, in one embodiment theinvention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4,or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or r. In somements, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 20-35 orgreater comprise at least three XTEN with each XTEN having at least 42 to about 288 amino acids andwherein at least two of the XTEN are linked to the fusion protein with no less than about 100, or about200, or about 300, or about 400, or about 500 amino acids of separation between the two XTEN. In otherembodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 20-35 orgreater comprise at least four XTEN with each XTEN having at least 42 to about 288 amino acids andwherein at least three of the XTEN are linked to the fusion protein with no less than about 100, or about200, or about 300, or about 400 amino acids of separation between any two of the three XTEN.
In another embodiment, the invention provides a CFXTEN in which the Ratio XTEN Radii isat least 20-35 or greater, the CFXTEN comprises at least three XTEN with each XTEN having at least42 to about 288 amino acids and n at least two of the three of the XTEN linked to the fusionprotein are ted by an amino acid sequence of at least 100, or about 200, or about 300 to about 400amino acids, and the third XTEN is linked within the B domain (or fragment thereof) or within the Cdomain (or the terminus thereof). In another embodiment, the invention provides a CFXTEN in whichthe Ratio XTEN Radii is at least 20-35 or greater, the CFXTEN ses at least four XTEN with eachXTEN having at least 42 to about 288 amino acids and wherein at least three of the four of the XTENlinked to the fusion protein are ted by an amino acid ce of at least 300 to about 400 aminoacids and the fourth XTEN is linked within the B domain (or fragment thereof) or within the C domain(or the terminus thereof).
In yet other embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii isat least 20-35 or greater, the CFXTEN comprises at least five XTEN with four XTEN having at least 42to about 144 amino acids wherein at least four of the XTEN are linked to the fusion protein with no lessthan about 100, 200, or about 300, or about 400 amino acids of separation between any two of the fourXTEN and a fifth XTEN is linked Within the B domain (or nt thereof) or Within the C domain (orthe terminus thereof). In one embodiment, the invention provides a CFXTEN in Which the Ratio XTENRadii is at least 20-35 or greater, the CFXTEN comprises at least five XTEN With four XTEN having atleast 42 to about 144 amino acids Wherein at least three of the XTEN linked to the fusion protein areted by an amino acid sequence of at least 300 to about 400 amino acids, the fourth XTEN is linkedWithin the B domain (or fragment thereof) and a fifth XTEN is linked Within the C domain (or theterminus thereof).
In one aspect, the invention provides CFXTEN in Which the Ratio XTEN Radii is at least 2.0,or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5or greater, and the composition does not comprise certain sequences. In one embodiment of theforegoing, the invention provides CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greaterWith the proviso that the fusion protein does not comprise a sequence from any one of Table 50 or Table51. In another embodiment of the foregoing, the ion provides CFXTEN in Which the Ratio XTENRadii is at least 20-35 or greater With the proviso that the fusion protein does not comprise a sequencehaVing an AG family XTEN ce. In r embodiment of the foregoing, the invention providesCFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusionprotein does not se a sequence selected from GTPGSGTASSSP (SEQ ID NO: 31),GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ IDNO: 34). In another embodiment of the foregoing, the invention es CFXTEN in Which the RatioXTEN Radii is at least 20-35 or greater With the proviso that the fusion protein does not se anyone of the sequences selected from TASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ IDNO: 32), STGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34) andGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing, the invention providesCFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusionprotein does not comprise a sequence selected fromGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59),PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS (SEQ ID NO: 71), orPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS (SEQ ID NO: 80). In another embodiment of the foregoing, the inventionprovides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the o that thefusion protein does not comprise an XTEN sequence ting ofGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP (SEQ ID NO: 59),PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS (SEQ ID NO: 71), orPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS (SEQ ID NO: 80).
In one aspect, the present invention provides methods to create CFXTEN with XTEN insertedto maximize the steric interference of FVIII binding agents that would otherwise bind to FVIII andneutralize procoagulant activity or result in the clearance or degradation of FVIII. Accordingly, in oneembodiment, the invention provides a method comprising the steps of selecting a FVIII sequence with atleast 90% sequence identity to a sequence of Table 1, selecting three or more XTEN from Table 4 inwhich the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or greater, creating sion constructs designed tolocate said XTEN at or proximal to locations selected from Table 5, Table 6, Table 7, Table 8, and Table9, wherein the three or more XTEN are at least 300 to 400 amino acids, expressing and recovering theresulting CFXTEN, and assaying the ing fusion proteins in an assay described herein in order toconfirm the reduced binding of the CFXTEN fusion protein. By the inventive method, a CFXTENexhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, orat least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or atleast 70% reduced, or at least 80% reduced binding to a FVIII binding agent including, but not limited tothe antibodies of Table 10, and ts procoagulant actiVity.
. CFXTEN Fusion Protein Configurations with Spacer and Cleavage Sequences] In another aspect, the invention provides CFXTEN red with one or more spacersequences orated into or adjacent to the XTEN that are designed to incorporate or enhance aonality or property to the composition, or as an aid in the assembly or manufacture of the fusionprotein itions. Such properties include, but are not limited to, inclusion of cleavage sequence(s)to permit release of components, inclusion of amino acids ible with nucleotide ctions sites topermit e of XTEN-encoding nucleotides to FVIII-encoding nucleotides or that facilitateconstruction of expression vectors, and linkers designed to reduce steric hindrance in s ofCFXTEN fusion proteins. 2012/046326] In an ment, a spacer sequence can be uced between an XTEN ce and aFVIII component to decrease steric hindrance such that the FVIII component may assume its dry structure and/or interact appropriately with its target substrate or processing enzyme. For spacersand methods of identifying desirable spacers, see, for example, , et al. (2003) Protein Engineering—879, specif1cally incorporated by reference herein. In one embodiment, the spacer comprises oneor more peptide sequences that are between 1—50 amino acid residues in length, or about 1—25 residues,or about 1-10 residues in length. Spacer sequences, ive of cleavage sites, can comprise any of thenatural L amino acids, and will preferably have XTEN-like properties in that the majority of residueswill be hydrophilic amino acids that are sterically ered such as, but not limited to, glycine (G),alanine (A), serine (S), threonine (T), glutamate (E), proline (P) and aspartate (D). The spacer can be asingle glycine residue, polyglycines or polyalanines, or is inately a mixture of combinations ofglycine, serine and alanine residues. In one embodiment, a spacer sequence, exclusive of cleavage siteamino acids, has about 1 to 10 amino acids that consist of amino acids selected from glycine (G), alanine(A), serine (S), threonine (T), ate (E), and proline (P) and are substantially devoid of secondarystructure; e. g., less than about 10%, or less than about 5% as determined by the Chou-Fasman and/orGOR algorithms. In one embodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In anotherembodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612) linked to a cleavage sequence ofTable 12. In addition, spacer sequences are designed to avoid the introduction of T-cell epitopes whichcan, in part, be achieved by avoiding or limiting the number of hydrophobic amino acids utilized in thespacer; the determination of epitopes is described above and in the Examples.
In a particular embodiment, the CFXTEN fusion protein comprises one or more spacersequences linked at the junction(s) between the payload FVIII sequence and the one or more XTENincorporated into the fusion protein, n the spacer sequences comprise amino acids that arecompatible with nucleotides encoding restriction sites. In r embodiment, the CFXTEN fusionn comprises one or more spacer sequences linked at the junction(s) between the payload FVIIIsequence and the one more XTEN incorporated into the fusion protein wherein the spacer sequencescomprise amino acids that are compatible with nucleotides encoding restriction sites and the amino acidsand the one more spacer sequence amino acids are chosen from glycine (G), alanine (A), serine (S),ine (T), glutamate (E), and proline (P). In another embodiment, the CFXTEN fusion proteincomprises one or more spacer sequences linked at the junction(s) between the payload FVIII sequenceand one more XTEN incorporated into the fusion protein wherein the spacer sequences comprise aminoacids that are compatible with nucleotides encoding ction sites and the one more spacer sequencesare chosen from the sequences of Table 11. The exact sequence of each spacer sequence is chosen to becompatible with cloning sites in expression s that are used for a particular CFXTEN construct. Inone embodiment, the spacer sequence has properties compatible with XTEN. In one embodiment, thespacer sequence is GAGSPGAETA (SEQ ID NO: 178). For XTEN sequences that are incorporatedinternal to the FVIII sequence, each XTEN would generally be flanked by two spacer sequencescomprising amino acids compatible with restriction sites, while XTEN attached to the N— or C-terminuswould only require a single spacer sequence at the junction of the two components and another at theopposite end for incorporation into the vector. As would be apparent to one of ordinary skill in the art,the spacer sequences comprising amino acids compatible with restriction sites that are internal to FVIIIcould be d from the construct when an entire CFXTEN gene is synthetically ted.
Table 11: Spacer Seguences Compatible with Restriction SitesSpacer Sequence Restriction EnzymeGSPG (SEQ ID NO: 174) BsaIETET (SEQ ID NO: 175) BsaIPGSSS (SEQ ID NO: 176) BbsIGAP AscIGPA FseIGPSGP (SEQ ID NO: 177) SfiIAAA SacIITG AgeIGT KpnIGAGSPGAETA (SEQ IDNO: 178) SfiIASS XhoI In another aspect, the present invention provides CFXTEN urations with cleavagesequences incorporated into the spacer sequences. In some embodiments, spacer sequences in aCFXTEN fusion protein composition comprise one or more cleavage sequences, which are identical ordifferent, wherein the cleavage ce may be acted on by a protease, as shown in , to releaseFVIII, a FVIII component (e. g., the B domain) or XTEN sequence(s) from the fusion protein. In oneembodiment, the incorporation of the cleavage sequence into the CFXTEN is ed to permit releaseof the FVIII component that becomes active or more active (with respect to its ability serve as amembrane binding site for factors IXa and X) upon its release from the XTEN. In the foregoingembodiment, the procoagulant activity of FVIII component of the CFXTEN is increased after cleavageby at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90% compared to the intact . The cleavage sequences are located sufficiently close to theFVIII sequences, lly within 18, or within 12, or within 6, or within 2 amino acids of the FVIIIsequence, such that any remaining residues attached to the FVIII after cleavage do not appreciablyinterfere with the activity (e.g., such as binding to a clotting n) of the FVIII, yet provide ientaccess to the protease to be able to effect cleavage of the cleavage sequence. In some cases, theCFXTEN sing the cleavage sequences will also have one or more spacer sequence amino acidsn the FVIII and the cleavage sequence or the XTEN and the cleavage sequence to facilitate accessof the protease; the spacer amino acids comprising any natural amino acid, including e, serine andalanine as red amino acids. In one embodiment, the cleavage site is a sequence that can be cleavedby a protease endogenous to the mammalian subject such that the CFXTEN can be cleaved afteradministration to a subject. In such case, the CFXTEN can serve as a prodrug or a circulating depot forthe FVIII. In a ular construct of the foregoing, the CFXTEN would have one or two XTEN linkedto the N— and/or the C-terminus of a BDD via a cleavage ce that can be acted upon by anactivated coagulation factor, and would have an additional XTEN located between the processing aminoacids at position R740 and R1689 such that the XTEN could be released, leaving a form of FVIII similarto native activated FVIII. In one embodiment of the foregoing construct, the FVIII that is released fromthe fusion protein by cleavage of the cleavage sequence exhibits at least about a two-fold, or at leastabout a three-fold, or at least about a four-fold, or at least about a five-fold, or at least about a six-fold, orat least about a eight-fold, or at least about a ten-fold, or at least about a 20-fold se in tycompared to the intact CFXTEN fusion protein.
Examples of cleavage sites contemplated by the ion e, but are not d to, apolypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FXIIa,kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), Elastase-2, granzyme B, MMP-l2, , MMP-l7or MMP-20, or by non-mammalian proteases such as TEV, enterokinase, PreScissionTM proteasevirus 3C protease), and sortase A. Sequences known to be cleaved by the foregoing proteases andothers are known in the art. Exemplary cleavage sequences contemplated by the invention and thetive cut sites within the sequences are presented in Table 12, as well as sequence variants thereof.
For CFXTEN sing incorporated cleavage sequence(s), it is generally preferred that the one ormore cleavage sequences are substrates for activated clotting proteins. For example, thrombin (activatedclotting factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N.D., et al. (2008)Nucleic Acids Res, 36: D320], which is cut after the arginine at position 4 in the sequence. Active FIIais produced by cleavage of FII by FXa in the ce of phospholipids and calcium and is down streamfrom factor VIII in the coagulation pathway. Once activated, its natural role in coagulation is to cleavefibrinogen, which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurswhen coagulation is necessary for proper hemostasis. By incorporation of the LTPRSLLV sequence(SEQ ID NO: 1618) into the CFXTEN between and linking the FVIII and the XTEN components, theXTEN is removed from the adjoining FVIII concurrent with activation of either the extrinsic or intrinsiccoagulation pathways when coagulation is required logically, thereby selectively ing FVIII.
In another embodiment, the invention provides CFXTEN with orated FXIa cleavage sequencesbetween the FVIII and XTEN component(s) that are acted upon only by initiation of the intrinsiccoagulation system, wherein a gulant form of FVIII is released from XTEN by FXIa to participatein the coagulation cascade. While not intending to be bound by any particular theory, it is believed thatthe CFXTEN of the foregoing embodiment would sequester the FVIII away from the other ationfactors except at the site of active clotting, thus allowing for larger doses (and therefore longer dosingintervals) with minimal safety concerns.
Thus, cleavage sequences, particularly those susceptible to the gulant activated clottingproteins listed in Table 12, would provide for ned release of FVIII that, in certain embodiments ofthe CFXTEN, can provide a higher degree of activity for the FVIII component released from the intactform of the CFXTEN, as well as additional safety margin for high doses of CFXTEN administered to asubject. In one embodiment, the ion provides CFXTEN comprising one or more gesequences operably oned to release the FVIII from the fusion protein upon cleavage, wherein theone or more cleavage sequences has at least about 86%, or at least about 92%, or 100% sequence identityto a sequence selected from Table 12.
In some embodiments, only the two or three amino acids flanking both sides of the cut site(four to six amino acids total) are incorporated into the cleavage sequence that, in turn, is incorporatedinto the CFXTEN of the embodiments, providing, e.g., XTEN release sites. In other embodiments, theincorporated cleavage sequence of Table 12 can have one or more deletions or insertions or one or two orthree amino acid substitutions for any one or two or three amino acids in the known ce, whereinthe deletions, insertions or substitutions result in reduced or enhanced tibility but not an e ofsusceptibility to the protease, resulting in an ability to tailor the rate of release of the FVIII from theXTEN. Exemplary substitutions within cleavage ces that are utilized in the CFXTEN of theinvention are shown in Table 12.
Table 12: Protease Cleavage SeguencesEggaggmjg @2312? 8&3; Minimalcmsne SENgDequenceFXIa KLTRtAET 179 KD/FL/T/RlVA/VE/GT/GVFXIa DFTRVVVG 18o KD/FL/T/RlVA/VE/GT/GVFXIIa VGG 181 NAKallikrein SPFRlSTGG 182 -/-/FL/RY¢SR/RT/—/—FVIIa LQVRVIVGG 183 NAFIXa PLGRVIVGG 184 -/-/G/R¢-/—/—/—FXa IEGRtTVGG 185 FP/RlSTI/VFSHGFIIa (thrombin) LTPRVSLLV 186 —/—/PLA/R¢SAG/—/—/—Elastase-2 LGPVVSGVP 187 —/—/—/v1ATt—/—/—/—me-B VAGDISLEE 188 V/-/-/Dt-/-/-/-MMP-12 GPAGVLGGA 189 G/PA/—/GtL/—/G/— 19oMMP-13 GPAGVLRGA 191 G/P/—/GtL/—/GA/— 192MMP-17 APLGVLRLR 193 -/PS/-/—¢LQ/—/LT/—MMP-2O PALPtLVAQ 194 NATEV ENLYFQtG 195 ENLYFQtG/s 196Enterokinase DDDKMVGG 197 DDDKMVGG 198(Pizgieizzieoigll) 200LEVLFQtGP 199 LEVLFQIGPSortase A LPKTtGSEs 201 L/P/KEAD/TlG/JEKS/S 202lindicates cleavage site NA: not applicablethe listing of multiple amino acids before, between, or after a slash te alternative aminoacids that can be substituted at the position; - indicates that any amino acid may besubstituted for the corresponding amino acid indicated in the middle column6. Exemplary CFXTEN Fusion Protein Sequences Non-limiting examples of sequences of fusion proteins containing a single FVIII linked to oneor more XTEN are presented in Table 21 . The exemplary amino acid sequences of Table 21 (and theDNA ces that encode them) contain his tags for purification purposes that, as would be apparent toone of skill in the art, can be deleted from the sequence without having an effect on the procoagulantactivity of the CFXTEN fusion protein. In one embodiment, the CFXTEN of Table 21 further compriseamino acids on the N—terminus corresponding to that of native human FVIII (namely, the sequenceTCFFLCLLRFCFS (SEQ ID NO: 1611)) to aid in the sion and secretion of the CFXTENfusion n. In one embodiment, a CFXTEN composition comprises a fusion protein haVing at leastabout 80% sequence ty compared to a CFXTEN from Table 21, alternatively at least about 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or about 100% sequence identity as compared to a CFXTEN from Table 21, when optimally aligned. Inanother embodiment, a CFXTEN ition comprises a fusion protein from Table 21 in which the C-terminal his-his-his-his-his-his sequence (SEQ ID NO: 1700) deleted. However, the invention alsocontemplates substitution of any of the FVIII sequences of Table l for a FVIII component of theCFXTEN of Table 21, and/or substitution of any sequence of any one of Tables 3, 4, and l3-l7 for anXTEN component of the CFXTEN of Table 21. Generally, the resulting CFXTEN of the foregoingexamples retain at least a portion of the gulant actiVity of the corresponding FVIII not linked to theXTEN. In the foregoing fusion proteins hereinabove described in this paragraph, the CFXTEN fusionprotein can further comprise one or more cleavage sequences; e.g., a ce from Table 12, thecleavage sequence being d between the FVIII and the XTEN sequences or between adjacent FVIIIdomains linked by XTEN. In some embodiments comprising cleavage sequence(s), the intact CFXTENcomposition has less actiVity but a longer half-life in its intact form compared to a ponding FVIIInot linked to the XTEN, but is designed such that upon administration to a t, the FVIII componentis gradually released from the fusion protein by cleavage at the cleavage sequence(s) by endogenousproteases, pon the FVIII component exhibits procoagulant activity.
The CFXTEN compositions of the embodiments can be evaluated for actiVity using assays orin vivo parameters as described herein (e. g., in vitro coagulation assays, assays of Table 49, or acodynamic effect in a preclinical hemophilia model or in clinical trials in humans, using methodsas described in the Examples or other methods known in the art for assessing FVIII actiVity) to determinethe suitability of the configuration or the FVIII sequence variant, and those CFXTEN compositions(including after cleavage of any incorporated XTEN-releasing cleavage sites) that retain at least about%, or about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about90%, or about 95% or more activity ed to native FVIII sequence are ered suitable for use inthe treatment of FVIII-related conditions.
V). PROPERTIES OF THE CFXTEN COMPOSITIONS OF THE INVENTION(a) Pharmacokinetic Properties of CFXTEN It is an object of the present invention to provide CFXTEN fusion proteins and pharmaceuticalcompositions comprising CFXTEN with ed pharmacokinetics compared to FVIII not linked toXTEN. The pharmacokinetic properties of a FVIII ed by linking a given XTEN to the FVIIIinclude, but are not limited to, terminal half-life, area under the curve (AUC), Cmax, volume ofdistribution, ining the biologically active CFXTEN above a minimum effective blood unitconcentration for a longer period of time compared to the FVIII not linked to XTEN. The enhancedproperties permit less frequent dosing and/or a longer-lived procoagulant effect compared to acomparable dose of FVIII not linked to XTEN. Enhancement of one or more of these ties canresulting benefits in the treatment of factor VIII-related conditions.
Exogenously administered factor VIII has been reported to have a al half-life in humansof approximately 12-14 hours when complexed with normal von Willebrand factor protein, whereas inthe absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham EG,et al., Br J Haematol. (1982) 52(2):259-267; Bjorkman, S., et al. Clin Pharmacokinet. (2001) 40:815). Asa result of the enhanced properties conferred by XTEN, the CFXTEN, when used at the dose and doseregimen ined to be appropriate for the subject and its underlying condition, can achieve acirculating concentration resulting in a desired procoagulant or clinical effect for an extended period oftime compared to a comparable dose of the ponding FVIII not linked to XTEN. As used herein, a“comparable dose” means a dose with an equivalent moles/kg or International Units/kg (IU/kg) for thecomposition that is administered to a t. It will be understood in the art that a ”comparable dose" ofFVIII not linked to XTEN would represent a lesser weight of drug but would have essentially the sameIUs or mole-equivalents of CFXTEN in the dose.
An international unit (“IU”) of factor VIII is defined in the art as the coagulant actiVity presentin 1 ml of normal human plasma. A normal, non-hemophilic individual human is expected to have about100 IU/dL factor VIII ty. In hemophilia A, the doses ed to treat are dependent on thecondition. For minor bleeding, doses of native or recombinant factor VIII of 20 to 40 IU/kg are typicallyadministered, as necessary. For moderate bleeding, doses of 30 to 60 IU/kg are administered asnecessary, and for major bleeding, doses of 80 to 100 IU/kg may be required, with repeat doses of 20 toIU/kg given every 8 to 12 hours until the bleeding is resolved. For prophylaxis against bleeding inpatients with severe hemophilia A, the usual doses of native or recombinant FVIII preparations are 20 to40 lU/kg body weight at intervals of about 2 to 3 days. A rd on for estimating an appropriatedose of a composition sing FVIII is:Required units = body weight (kg) x desired factor VIII rise (IU/dL or % of normal) x 0.5(IU/kg per IU/dL).
In many cases, the therapeutic levels for FVIII in subjects of different ages or degree of diseasehave been ished and are available in published literature or are stated on the drug label for approvedproducts ning the FVIII. For example, the Subcommittee on Factor VIII and Factor IX of theific and Standardization Committee of the International Society on Thrombosis and Haemostasisposted, on the ISTH Website 29 November, 2000, that the most widely used measure of hemophilia A isestablished by determining the circulating trations of plasma FVIII procoagulant levels, withpersons with <l% (< 0.01 IU/ml) factor VIII defined as severe; 1-5% (0.01 - 0.05 IU/ml) as moderatelysevere; and >5-40% (0.05 - <0.40 IU/ml) as mild, where normal is 1 IU/ml of factor VIIIC (100%). Thetherapeutic levels can be established for new compositions, including those CFXTEN and pharmaceuticalcompositions comprising CFXTEN of the disclosure, using standard methods. In practicing the presentinvention, it will be tood that any dosage of CFXTEN that is effective may be used for treatingbleeding episodes or maintaining asis. The methods for establishing the therapeutic levels anddosing les for a given composition are known to those of skill in the art (see, e. g., n &'s The Pharmacological Basis of Therapeutics, 11th Edition, McGraw—Hill (2005)). For example,by using dose-escalation studies in subjects with the target condition to determine efficacy or a desirablecologic effect, ance of adverse events, and determination of circulating blood levels, theeutic blood levels for a given subject or population of subjects can be determined for a given drugor biologic. The dose escalation studies would evaluate the activity of a CFXTEN through studies in asubject or group of hemophilia A subjects. The studies would monitor blood levels of procoagulant, aswell as physiological or clinical parameters as known in the art or as described herein for one or moreparameters associated with the factor VIII-related condition, or clinical parameters ated with abeneficial outcome, together with observations and/or measured parameters to determine the no effectdose, adverse events, minimum effective dose and the like, together with measurement ofpharmacokinetic parameters that ish the determined or d circulating blood levels. The resultscan then be correlated with the dose administered and the blood concentrations of the therapeutic that arecoincident with the foregoing determined parameters or effect levels. By these methods, a range of dosesand blood concentrations can be correlated to the m effective dose as well as the maximum doseand blood concentration at which a desired effect occurs or is maintained and the period for which it canbe maintained, thereby establishing the therapeutic blood levels and dosing schedule for the composition.
Thus, by the foregoing methods, a Cmin blood level is established, below which the CFXTEN fusionprotein would not have the desired pharmacologic effect and a Cmax blood level, above which side effectssuch as thrombosis may occur (Brobrow, RS, JABFP (2005) 18(2):]47-149), establishing the therapeuticwindow for the composition.
One of skill in the art can, by the means disclosed herein or by other methods known in the art,confirm that the administered CFXTEN remains at therapeutic blood levels to in hemostasis forthe desired interval or requires adjustment in dose or length or sequence of XTEN. Further, thedetermination of the appropriate dose and dose frequency to keep the CFXTEN within the therapeuticwindow establishes the therapeutically effective dose regimen; the schedule for stration ofmultiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in needf resulting in consecutive Cmax peaks and/or Cmin troughs that remain above eutically-effective concentrations and result in an improvement in at least one measured parameter relevant for thetarget condition. In one embodiment, the CFXTEN or a pharmaceutical compositions comprisingCFXTEN administered at an appropriate dose to a subject results in blood concentrations of theCFXTEN fusion protein that remains above the minimum ive concentration to maintain hemostasisfor a period at least about two-fold longer compared to the corresponding FVIII not linked to XTEN andadministered at a comparable dose; alternatively at least about three-fold longer; alternatively at leastabout four-fold longer; alternatively at least about five-fold longer; atively at least about siX-foldlonger; alternatively at least about seven-fold longer; alternatively at least about eight-fold ;alternatively at least about nine-fold longer, alternatively at least about ld longer, or at least abouttwenty-fold longer or greater ed to the corresponding FVIII not linked to XTEN and administeredat a comparable dose. As used herein, an “appropriate dose” means a dose of a drug or biologic that,when administered to a subject, would result in a desirable therapeutic or pharmacologic effect (e.g.,asis) and/or a blood concentration within the therapeutic window.
] In practicing the invention, CFXTEN with longer terminal half-life are generally preferred, soas to improve patient convenience, to increase the interval between doses and to reduce the amount ofdrug ed to achieve a sustained effect. The enhanced PK parameters allow for reduced dosing of thesubject compositions, compared to FVIII not linked to XTEN, particularly for those hemophilia Asubjects receiving e prophylaxis.
As described more fully in the Examples pertaining to pharmacokinetic characteristics of fusionproteins comprising XTEN, it was observed that increasing the total length of the XTEN, singly or incombination, confers a disproportionate increase in the terminal half-life of a fusion protein comprisingthe XTEN. Accordingly, the invention provides CFXTEN fusion proteins and pharmaceuticalitions comprising CFXTEN wherein the CFXTEN exhibits an enhanced half-life whenadministered to a t. In some embodiments, the invention provides monomeric CFXTEN fusionproteins sing one or more XTEN wherein the number and location of the XTEN are selected toconfer an increase in the terminal half-life for the CFXTEN administered to a subject compared to thecorresponding FVIII not linked to the XTEN and administered at a able dose, wherein theincrease is at least about two-fold longer, or at least about three-fold, or at least about four-fold, or atleast about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, orat least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at leasta 40-fold or greater increase in terminal ife compared to the FVIII not linked to the XTEN. In otherembodiments, the invention provides CXTEN compositions and pharmaceutical compositionscomprising CFXTEN wherein the stration of a composition to a subject in need thereof results in aterminal half-life that is at least 12 h greater, or at least about 24 h greater, or at least about 48 h r,or at least about 96 h greater, or at least about 144 h greater, or at least about 7 days r, or at leastabout 14 days greater, or at least about 21 days greater compared to a comparable dose of FVIII notlinked to XTEN. In another embodiment, administration of a coagulation-effective dose of a CFXTENfusion protein to a subject in need f can result in a gain in time between utive dosesary to maintain blood levels of about 0.1 IU/ml of at least 48 h, or at least 72 h, or at least about96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 daysbetween consecutive doses compared to a FVIII not linked to XTEN and administered at a comparabledose.
In one embodiment, the present invention provides CFXTEN fusion proteins andpharmaceutical compositions comprising CFXTEN that exhibit, when administered to a subject in needthereof, an increase in AUC of at least about 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%, or at least about a 100%, or at least about 150%, or at least about200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about a 2000%compared to the corresponding FVIII not linked to the XTEN and administered to a subject at acomparable dose. The pharmacokinetic parameters of a CFXTEN can be determined by rdmethods involving , the taking of blood samples at timed intervals, and the assaying of the proteinusing ELISA, HPLC, radioassay, clotting , the assays of Table 49, or other methods known in theart or as described herein, followed by standard calculations of the data to derive the half-life and otherPK ters.
In one embodiment, a smaller IU amount of about two-fold less, or about three-fold less, orabout four-fold less, or about five-fold less, or about siX-fold less, or about eight-fold less, or about 10-fold less or greater of the fusion protein is administered in ison to the corresponding FVIII notlinked to the XTEN under a dose n needed to maintain hemostasis and the fusion protein achievesa comparable area under the curve as the corresponding IU amount of the FVIII not linked to the XTENneeded to in hemostasis. In another embodiment, the CFXTEN fusion n or a pharmaceuticalitions comprising CFXTEN requires less frequent administration for routine laxis of ahemophilia A subject, wherein the dose of fusion protein is administered about every four days, aboutevery seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly to thet, and the fusion protein achieves a comparable area under the curve as the corresponding FVIIInot linked to the XTEN and administered to the subject. In yet other embodiments, an accumulativesmaller IU amount of about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90% less of the fusion n is administered to a subject incomparison to the corresponding IU amount of the FVIII not linked to the XTEN under a dose regimenneeded to maintain a blood concentration of 0.1 IU/ml, yet the fusion protein achieves at least acomparable area under the curve as the corresponding FVIII not linked to the XTEN. The accumulativesmaller IU amount is measure for a period of at least about one week, or about 14 days, or about 21 days,or about one month.
In one aspect, the invention provides CFXTEN compositions designed to reduce binding byFVIII binding agents, thereby increasing the al half-life of CFXTEN administered to a subject,while still retaining procoagulant activity. It is believed that the CFXTEN of the present invention havecomparatively higher and/or ned activity achieved by reduced active clearance of the molecule bythe addition of unstructured XTEN to the FVIII coagulation . The clearance mechanisms toremove FVIII from the circulation have yet to be fully elucidated. Uptake, elimination, and inactivationof coagulation proteins can occur in the circulatory system as well as in the extravascular space.
Coagulation factors are complex proteins that interact with a large number of other proteins, lipids, andreceptors, and many of these ctions can contribute to the elimination of CFs from the circulation.
The protein von rand factor is an example of a FVIII binding agent that binds to FVIII. FactorVIII and von Willebrand factor (VWF) circulate in the blood as a tight, non-covalently linked complex inwhich VWF serves as a carrier that likely butes to the protection of FVIII from active cleavagemechanisms, yet nevertheless s in a limitation on the terminal ife of FVIII. For example: (i)VWF stabilizes the heterodimeric structure of FVIII; (ii) VWF protects FVIII from lyticdegradation by phospholipid-binding proteases like activated protein C and activated FX (FXa); (iii)VWF interferes with binding of FVIII to negatively charged phospholipid surfaces exposed withinactivated platelets; (iV) VWF inhibits binding of FVIII to activated FIX (FIXa), thereby denying FVIIIaccess to the FX-activating complex; and (V) VWF prevents the cellular uptake of FVIII (Lenting, P.J., etal., J osis and Haemostasis (2007) 5(7):1353-1360). In on, LDL receptor-related n(LRPl, also known as crogobulin receptor or CD91) has been identified as a candidate clearancereceptor for FVIII, with LRPl binding sites identified on both chains of the heterodimer form of FVIII(Lenting PJ, et al.,. J Biol Chem (1999) 274: 23734—23739; Saenko EL, et al., J Biol Chem (1999) 274:37685—37692). LRPs are involved in the clearance of a diversity of ligands including proteases,inhibitors of the Kunitz type, protease serpin complexes, lipases and lipoproteins (Narita, et al., Blood(1998) 2:555-560). It has been shown that the light chain, but not the heavy chain, of factor VIII binds tosurface-exposed LRPl receptor protein (Lentig et al. (J Biol Chem (1999) 274(34):23734-23739; andUS. Pat. No. 6,919,311), which suggests that LRPl may play an essential role in the active clearance ofproteins like FVIII. While the VWF—FVIII interaction is of high y (<1 nM), the complex isnevertheless in a dynamic equilibrium, such that a small but significant portion of the FVIII molecules(5—8%) ate as a free protein (Leyte A, et al., m J (1989) 257: 679—683; Noe DA.
Haemostasis (1996) 26: 289—3 03). As such, a portion of native FVIII is unprotected by VWF, allowingactive clearance mechanisms to remove the unprotected FVIII from the circulation.
In one embodiment, the invention provides CFXTEN that associate with VWF but haveenhanced protection from active clearance receptors conferred by the incorporation of two more XTENat one or more locations within the FVIII molecule (e. g., ons selected from Table 5, Table 6, Table7, Table 8, and Table 9 or FIGS. 8-9), wherein the XTEN interfere with the interaction of the resultingCFXTEN with those clearance receptors with the result that the pharmacokinetic properties of theCFXTEN is enhanced compared to the corresponding FVIII not linked to XTEN. In anotherembodiment, the invention provides CFXTEN that have reduced binding affinity with VWF of at least% less, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70% less, butare nevertheless configured to have enhanced protection from active clearance ors conferred by theincorporation ofXTEN at one or more locations within the FVIII molecule, wherein the XTEN interferewith the interaction of factor VIII with those receptors. In the foregoing embodiments, the CFXTENhave an increased terminal half-life of at least about 12 h, or 24 h, or 48 h, or 72 h, or 96 h, or 120 h, or144 h, or 7 days, or 10 days, or 14 days, or 21 days compared to the FVIII not linked to XTEN. Theinvention provides a method to create CFXTEN with reduced clearance n the CFXTEN fusionproteins created with the multiple insertions are ted for inhibition of binding to clearance receptors,compared to FVIII not linked to XTEN, using in vitro binding assays or in vivo pharmacokinetic modelsdescribed herein or other assays known in the art, and selecting those that demonstrate reduced bindingyet retain procoagulant FVIII actiVity. In addition, the foregoing fusion proteins can be optimized tohave increased Ratio XTEN Radii of at least 2.0-3.5 in order to achieve pharmacokinetic properties thatare further enhanced. Table 5, Table 6, Table 7, Table 8, and Table 9 and FIGS. 8-9 provide non-limitingexamples ofXTEN insertion points within the factor VIII sequence. Using such insertion points, theinvention contemplates CFXTEN compositions that have configurations with multiple XTEN insertedwith about 100, or about 200, or about 300, or about 400, or about 500 amino acids separating at leastthree XTEN to further increase the protection against active nce mechanisms and, hence, increasethe terminal half-life of the CFXTEN. Not to be bound by a particular theory, the XTEN of the CFXTENcompositions with high net charge (e.g., CFXTEN comprising AE family XTEN) are expected, asdescribed above, to have less non-specific interactions with various negatively-charged surfaces such asblood vessels, tissues, or various receptors, which would r bute to reduced active clearance.
Conversely, the XTEN of the CFXTEN compositions with a low (or no) net charge (e. g., CFXTENcomprising AG family XTEN) are expected to have a higher degree of interaction with surfaces that,while contributing to active clearance, can potentiate the ty of the ated coagulation factor,given the known contribution of cell (e.g., platelets) and vascular es to the coagulation process andthe intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973;London, F., et al. Biochemistry (2000) 39(32):9850—985 8). The invention, in part, takes advantage of thefact that certain ligands wherein reduced g to a nce receptor, either as a result of a sedon—rate or an increased off-rate, may be effected by the obstruction of a or site by an insertedXTEN forming random coil, resulting in the d binding. The choice of the particular configurationof the CFXTEN fusion protein can be tested by methods disclosed herein to confirm those configurationsthat reduce the degree of binding to a clearance receptor such that a reduced rate of active nce isachieved. In one embodiment, the CFXTEN comprises a FVIII-XTEN sequence that has one or moreXTEN ed at locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9wherein the al half-life of the CFXTEN is increased at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least abouteight-fold, or at least about ten-fold, or at least about twenty-fold compared to a FVIII not linked to anXTEN. In another embodiment, the CFXTEN comprises a FVIII-XTEN sequence that has a first and atleast a second XTEN inserted at a first and second location ed from Table 5, Table 6, Table 7,Table 8, and Table 9 or FIGS. 8-9 wherein the terminal half-life of the CFXTEN is increased at leastabout two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or atleast about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about twenty-foldcompared to a FVIII not linked to an XTEN. In yet another ment, the CFXTEN comprises aFVIII-XTEN sequence that orates multiple XTEN sequences using three of more XTEN insertionlocations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 separated by about100, or about 200, or about 300, or about 400, or about 500 amino acids, n the terminal half-life ofthe CFXTEN is increased at least about two-fold, or at least about fold, or at least about four-fold,or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold,or at least about twenty-fold compared to a FVIII not linked to an XTEN. In the foregoing embodimentshereinabove described in this paragraph, the XTEN orated into the CFXTEN urations can beidentical or they can be different, and can have at least about 80%, or 90%, or 91%, or 92%, or 93%, or94%, or 95%, or 96%, or 97%, or 98%, or 99%, sequence identity to a sequence from any one of Tables3, 4, and 13-17, and can optionally include one or more cleavage sequences from Table 12, facilitatingrelease of one or more of the XTEN from the CFXTEN fusion protein.
In one embodiment, the invention provides CFXTEN that e the pharmacokinetics of thefusion protein by linking one or more XTEN to the FVIII component of the fusion protein n thefusion protein has an increase in apparent molecular weight factor of at least about two-fold, or at leastabout three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or atleast about seven-fold, or at least about fold, or at least about ten-fold, or at least about twelve-fold,or at least about -fold, and wherein the terminal half-life of the CFXTEN when administered to at is increased at least about two-fold, or at least about four-fold, or at least about eight-fold, or atleast about 10-fold or more compared to the corresponding FVIII not linked to XTEN. In the foregoingembodiment, wherein at least two XTEN molecules are orated into the CFXTEN, the XTEN canbe identical or they can be of a different sequence composition, net charge, or length. The XTEN canhave at least about 80%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or99%, sequence identity to a sequence from any one of Tables 3, 4, and 13-17, and can optionally includeone or more cleavage sequences from Table 12, facilitating release of one or more of the XTEN from theCFXTEN fusion protein.
] Thus, the invention es CFXTEN compositions in which the degree of activity,bioavailability, half-life or ochemical characteristic of the fusion protein can be tailored by theselection and placement of the type and length of the XTEN in the CFXTEN compositions. Accordingly,the invention contemplates compositions in which a FVIII from Table 1 and XTEN or XTEN fragmentfrom any one of Tables 3, 4, or 13-17 are produced, for example, in a configuration selected from anyone of formulae I-VIII or the XTEN are inserted at locations ed from Table 5, Table 6, Table 7,Table 8, and Table 9 or FIGS. 8-9 such that the construct has the desired property.
The invention provides methods to produce the CFXTEN compositions that can maintain theFVIII component at therapeutic levels in a subject in need f for at least a two-fold, or at least athree-fold, or at least a old, or at least a five-fold greater period of time compared to comparabledosages of the corresponding FVIII not linked to XTEN. In one embodiment of the method, the subjectis receiving routine prophylaxis to prevent bleeding episodes. In another embodiment of the method, thesubject is receiving treatment for a bleeding episode. In another embodiment of the method, the subject 2012/046326is receiving treatment to raise the circulating blood concentration of procoagulant FVIII above 1%, orabove 1-5%, or above 5-40% relative to FVIII concentrations in normal plasma. “Procoagulant” as usedherein has its general meaning in the art and generally refers to an ty that promotes clot formation,either in an in vitro assay or in vivo. The method to produce the compositions that can maintain the FVIIIent at eutic levels includes the steps of selecting one or more XTEN riate forconjugation to a FVIII to provide the desired pharmacokinetic properties in View of a given dose anddose regimen, creating a gene construct that encodes the CFXTEN in one of the configurations disclosedherein, transforming an riate host cell with an sion vector comprising the encoding gene,expressing the fusion protein under suitable culture conditions, recovering the CFXTEN, administrationof the CFXTEN to a mammal followed by assays to verify the pharmacokinetic properties and theactiVity of the CFXTEN fusion protein (e.g., the ability to maintain hemostasis or serve as aprocoagulant) and the safety of the administered composition. Those compositions exhibiting the desiredproperties are selected for further use. CFXTEN created by the methods provided herein can result inincreased efficacy of the stered composition by, amongst other properties, maintaining thecirculating concentrations of the procoagulant FVIII component at eutic levels for an enhancedperiod of time.
The invention provides methods to assay the CFXTEN fusion proteins of differing compositionor configuration in order to provide CFXTEN with the desired degree of gulant and therapeuticactivity and pharmacokinetic properties, as well as a ent safety profile. Specific in vitro and invivo assays or animal models are used to assess the activity and functional characteristics of eachconfigured CFXTEN and/or FVHI ent to be incorporated into CFXTEN, including but notlimited to the assays of the Examples, those assays of Table 49, as well as the following assays or othersuch assays known in the art for assaying the properties and effects of FVHI. Functional assays can beconducted that allow determination of ation activity, such as age clotting assay and two-stage ng assay wcliffe TW, Semin Thromb Hemost. (2002) 28(3):247-256), activated partialprothrombin (aPTT) assays (Belaaouaj AA et al., J. Biol. Chem. (2000) 275:27123-8; ollier JA.
Haemost (1994) 71 :339-46), chromogenic FVIH assays (Lethagen, S., et al., Scandinavian JHaematology (1986) 37:448—453), or animal model pharmacodynamic assays including bleeding time orthrombelastography (TEG or ROTEM), among others. Other assays include determining the bindingaffinity of a CFXTEN for the target substrate using binding or competitive binding assays, such asBiacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in US Patent,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in theart. Other assays to determine the binding of FVIH tors to CFXTEN include the Bethesda assay orthe Nijmegen modification of the Bethesda assay. The foregoing assays can also be used to assess FVHIsequence variants (assayed as single components or as CFXTEN fusion proteins) and can be compared tothe native FVIII to determine whether they have the same degree of procoagulant actiVity as the nativeCF, or some fraction thereof such that they are suitable for inclusion in CFXTEN; e. g., at least about%, or at least about 20$, or about 30%, or at least about 40%, or at least about 50%, or at least about60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to thenative FVIH.
Dose optimization is important for all drugs. A therapeutically effective dose or amount of theCFXTEN varies according to factors such as the disease state, age, seX, and weight of the individual, andthe ability of the stered fusion protein to elicit a desired response in the individual. For example, astandardized single dose of FVIII for all patients presenting with diverse bleeding conditions or abnormalclinical parameters (e.g., neutralizing antibodies) may not always be ive. Hemophilia A patientswith trauma, who have undergone surgery, or that have high titers of FVIII inhibitory antibodiesgenerally will require higher and more frequent dosing. Generally, dosage level is adjusted in frequency,duration, and units in keeping with the ty and duration of each patient's bleeding episode.ingly, the CFXTEN is included in the pharmaceutically acceptable carrier, delivery vehicle, orstabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the fusionprotein to stop bleeding, as measured by standard clotting assays. A consideration of these factors is wellwithin the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically orpharmacologically effective amount of the CFXTEN and the appropriated dosing schedule, versus thatamount that would result in insufficient potency such that clinical improvement or the arrest of bleedingis not achieved.
The invention provides methods to establish a dose regimen for the CFXTEN pharmaceuticalcompositions of the invention. The methods include administration of consecutive doses of atherapeutically effective amount of the CFXTEN pharmaceutical composition using variable periods oftime between doses to determine that interval of dosing ent to achieve and/or maintain the desiredparameter, blood level or al effect; such consecutive doses of a therapeutically ive amount atthe effective interval establishes the therapeutically effective dose regimen for the CFXTEN for a factorVIII-related disease state or condition. A prophylactically effective amount refers to an amount ofCFXTEN required for the period of time necessary to prevent a physiologic or clinical result or event;e.g., delayed onset of a ng episode or maintaining blood concentrations of procoagulant FVIH orequivalent above a threshold level (e. g., 1-5% to 5-40% of ). In the methods of treatment, thedosage amount of the CFXTEN that is administered to a subject ranges from about 5 to 300 dose,or from about 10 to 100 lU/kg/dose, or from about 20 to about 65 lU/kg/dose, or from about 20 to about40 lU/kg/dose for a subject. A suitable dosage may also depend on other factors that may influence theresponse to the drug; e. g., bleeding episodes generally requiring higher doses at more nt intervalscompared to prophylaxis.
In some ments, the method comprises administering a therapeutically-effective amountof a pharmaceutical composition comprising a CFXTEN fusion protein composition and at least onepharmaceutically acceptable carrier to a subject in need thereof, wherein the stration results in agreater improvement in at least one parameter or physiologic condition associated with a FVIIIdeficiency or coagulopathy, or s in a more favorable clinical outcome mediated by the FVIHcomponent of the CFXTEN compared to the effect on the parameter, ion or clinical outcomemediated by administration of a pharmaceutical composition comprising a FVIII not linked to XTEN andadministered at a comparable dose. Non-limiting examples of parameters that are improved includeblood concentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, areduced one-stage or two-stage ng assay time, delayed onset of a bleeding episode, a reducedchromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reducedBethesda titer to the CFXTEN relative to native FVIII. In one embodiment of the foregoing, theimprovement is achieved by administration of the CFXTEN pharmaceutical composition at a dose thates a circulating concentration of procoagulant FVIII (or equivalent) above a threshold level (e.g.,1-5% to 5-40% of normal FVIII levels), thereby establishing the therapeutically effective dose. Inanother embodiment of the ing, the improvement is achieved by administration of multipleutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dosen that maintains a circulating concentration of procoagulant FVIII (or equivalent) above athreshold level (e.g., 1-5% to 5-40% of normal FVIII ) for the length of the dosing period. Inanother embodiment of the method, the stration of at least two consecutive doses of the CFXTENpharmaceutical composition using a therapeutically effective dose regimen maintains a circulatingconcentration of procoagulant FVIII (or equivalent) above about 1%,, 2%, 3%, 4%, 5%, 10%, 15%, 20%,%, or 40% of normal FVIII levels for a period that is at least about three-fold longer; alternatively atleast about four-fold longer; alternatively at least about ld longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer;atively at least about nine-fold longer or at least about ten-fold longer compared to a FVIII notlinked to XTEN and stered using a therapeutically effective dose regimen In one ment, the CFXTEN or a pharmaceutical compositions comprising CFXTENadministered at a therapeutically ive dose regimen results in a gain in time of at least about three-fold longer; alternatively at least about four-fold longer; atively at least about five-fold longer;alternatively at least about siX-fold longer; alternatively at least about seven-fold longer; alternatively atleast about eight-fold longer; alternatively at least about old longer or at least about ten-fold longerbetween at least two consecutive Cmax peaks and/or Cmin troughs for blood levels of the fusion proteincompared to the corresponding biologically active protein of the fusion protein not linked to the XTENand administered at a comparable dose regimen to a subject. In another embodiment, the CFXTENadministered at a eutically effective dose regimen results in a comparable ement in one, ortwo, or three or more measured ters using less frequent dosing or a lower total dosage in IUs ofthe fusion protein of the pharmaceutical composition compared to the corresponding biologically activeprotein component(s) not linked to the XTEN and administered to a subject using a therapeuticallyive dose regimen for the FVIII. The measured parameters include any of the clinical, biochemical,or physiological parameters disclosed herein, or others known in the art for assessing subjects with factorVIII-related conditions.(b) Pharmacology and Pharmaceutical Properties of CFXTEN The present invention provides CFXTEN itions comprising FVIII covalently linked toXTEN that have enhanced pharmaceutical and pharmacology properties compared to FVIII not linked toXTEN, as well as methods to enhance the therapeutic and/or gulant effect of the FVIIIcomponents of the compositions. In addition, the invention provides CFXTEN compositions withenhanced properties compared to those art-known fusion proteins of factor VIII containing albumin,immunoglobulin polypeptide partners, polypeptides of shorter length and/or polypeptide partners withrepetitive sequences. In addition, CFXTEN fusion proteins e cant advantages over chemicalconjugates, such as pegylated constructs of FVIII, notably the fact that recombinant CFXTEN fusionproteins can be made in host cell expression systems, which can reduce time and cost at both the researchand development and manufacturing stages of a product, as well as result in a more homogeneous,defined product with less toxicity from both the t and metabolites of the CFXTEN compared topegylated conjugates.
As therapeutic agents, the CFXTEN possesses a number of advantages over therapeutics notcomprising XTEN, including one or more of the following non-limiting properties: increased solubility,increased thermal stability, reduced immunogenicity, increased apparent molecular weight, reduced renalclearance, reduced proteolysis, reduced metabolism, enhanced therapeutic efficiency, less frequentdosage regimen with increased time between doses capable of maintaining hemostasis in a subject withilia A, the ability to administer the CFXTEN composition subcutaneously or intramuscularly, a“tailored” rate of absorption when stered subcutaneously or intramuscularly, enhancedlyophilization stability, enhanced serum/plasma stability, increased terminal half-life, sed solubilityin blood stream, decreased binding by lizing dies, decreased active clearance, tailoredsubstrate g affinity, stability to degradation, stability to freeze-thaw, ity to proteases, stabilityto ubiquitination, ease of administration, compatibility with other pharmaceutical excipients or rs,persistence in the subject, increased stability in storage (e. g., increased shelf-life), and the like. The neteffect of the enhanced ties is that the use of a CFXTEN composition can result in an overallenhanced therapeutic effect compared to a FVIII not linked to XTEN, result in economic benefitsassociated with less frequent , and/or result in improved patient compliance when stered toa subject with a factor VIII-related condition.
The invention provides CFXTEN compositions and pharmaceutical compositions comprisingCFXTEN wherein the administration of the composition results in an improvement in at least one of theclinical or biochemical parameters disclosed herein as being useful for assessing the subject diseases,conditions or disorders. Non-limiting examples of parameters that are ed include bloodconcentrations of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, areduced one-stage or age clotting assay time, delayed onset of a bleeding episode, a reducedchromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reducedBethesda titer to the CFXTEN ve to native FVIII. The enhanced pharmacokinetic properties of thesubject CFXTEN s using an accumulatively lower IU dose of fusion protein to in theparameter ed to the corresponding FVIII component not linked to the XTEN. In onement, the total dose in IUs of an CFXTEN of the embodiments needed to achieve and maintainthe improvement in the at least one parameter for about 2-7 days is at least about three-fold lower, or atleast about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, orat least about 10-fold lower compared to the corresponding FVIII component not linked to the XTEN. Inanother embodiment, the total dose in IUs of a subject CFXTEN needed to achieve and maintain theimprovement in the at least one parameter over two, three or four consecutive doses is at least aboutthree-fold lower, or at least about four-fold, or at least about five-fold, or at least about six-fold, or atleast about eight-fold, or at least about d lower compared to the corresponding FVIII componentnot linked to the XTEN. Alternatively, the invention provides certain embodiments of CFXTEN whereinthe period between consecutive strations that results in achieving and ining theimprovement in at least one parameter is at least about three-fold, or at least about four-fold, or at leastabout five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10-fold longered to the corresponding FVIII component not linked to the XTEN and administered at acomparable IU dose. Alternatively, the invention provides n embodiments of CFXTEN whereinstration of 25 IU/kg results in a 30% improvement in a aPTT assay (or similar coagulation assay)time in a hemophilia A subject compared to 25 IU/kg of the corresponding FVIII not linked to XTENwhen assayed at about 2-7 days after administration. In yet another embodiment, the invention providesCFXTEN n administration of 25 IU/kg results in a 30% improvement in a bleeding time assaytime in a hemophilia A subject compared to 25 IU/kg of the ponding FVIII not linked to XTENwhen assayed at about 2-7 days after administration.
] In one embodiment, XTEN as a fusion partner increases the solubility of the FVIII payload.
Accordingly, where enhancement of the pharmaceutical or physicochemical properties of the FVIII isble, such as the degree of aqueous solubility or stability, the length and/or the motif familycomposition of the XTEN sequences incorporated into the fusion protein may each be selected to confera different degree of solubility and/or stability on the respective fusion proteins such that the overallpharmaceutical properties of the CFXTEN composition are enhanced. The CFXTEN fusion proteins canbe constructed and assayed, using methods described , to confirm the physicochemical propertiesand the choice of the XTEN length sequence or location adjusted, as needed, to result in the dproperties. In one embodiment, the CFXTEN has an aqueous solubility that is at least about 25% greatercompared to a FVIII not linked to the XTEN, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%,or at least about 400%, or at least about 500%, or at least about 1000% greater than the correspondingFVIII not linked to XTEN.
The invention es methods to produce and recover expressed CFXTEN from a host cellwith enhanced solubility and ease of recovery compared to FVIII not linked to XTEN. In oneembodiment, the method includes the steps of transforming a eukaryotic host cell with a cleotideencoding a CFXTEN with one or more XTEN components of cumulative sequence length greater thanabout 100, or greater than about 200, or greater than about 400, or r than about 600, or greater thanabout 800, or greater than about 1000, or greater than about 2000, or greater than about 3000 amino acidresidues, expressing the CFXTEN fusion protein in the host cell under suitable culture and inductionconditions, and recovering the expressed fusion protein in soluble form. In one embodiment, the one ormore XTEN of the CFXTEN fusion proteins each have at least about 80% sequence identity, or about90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTENselected from any one of Tables 4, and 13-17, or fragments thereof, and the FVIII have at least about80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% sequence identity compared toa FVIII selected from Table 1, and the CFXTEN components are in an N— to C-terminus configurationselected from any one of the uration embodiments disclosed herein.
VI). USES OF THE CFXTEN COMPOSITIONS The invention provides methods and ns for achieving a beneficial effect in a factor VIII-related condition by the administration of compositions comprising . As used herein, “factorVIII-related condition” is intended to include, but is not limited to factor VIII def1ciencies, ngdisorders related to factor VIII deficiency, hemophilia A, neutralization of factor VIII by anti-FVIIIantibodies or other factor VIII tors, and bleeding episodes resulting from trauma or surgery orvascular injury and other such conditions that can be ameliorated or corrected by administration of FVIIIto a subject. The inventive s achieve a beneficial effect while addressing disadvantages and/orlimitations of other s of treatment using factor VIII ations that have a relatively shortterminal half-life, require frequent administrations, are neutralized by inhibitors or have unfavorablepharmacoeconomics.
Hemostasis is ted by multiple protein factors, and such proteins, as well as analoguesthereof, have found utility in the treatment of factor VIII-related conditions. However, the use ofcommercially-available FVIII has met with less than optimal success in the management of subjectsafflicted with such conditions. In particular, dose optimization and frequency of dosing is important forFVIII used in maintaining circulating FVIII concentrations above threshold levels needed for hemostasis,as well as the treatment or prevention of bleeding episodes in hemophilia A subjects. The fact thatcommercially-available FVIII ts have a short ife necessitates nt dosing in order toachieve clinical benefit, which results in difficulties in the ment of such patients.
As established by the Subcommittee on Factor VIII and Factor IX of the Scientific andrdization Committee of the ational Society on Thrombosis and Haemostasis (posted on theISTH Website 29 November, 2000), the most widely used measure of the severity of hemophilia A isestablished by determining the circulating concentrations of plasma FVIII procoagulant levels, withs with <1% (< 0.01 IU/ml) factor VIII defined as severe; 1-5% (0.01 - 0.05 IU/ml) as moderatelysevere; and >5-40% (0.05 - <0.40 IU/ml) as mild, where normal is 1 IU/ml of factor VIIIC (100%).
The invention provides methods of treating a subject suffering from or at risk of developing afactor VIII-related condition. More particularly, the invention provides methods for treating orting controlling bleeding in subject. The subject can be any animal but preferably is a human. Inone embodiment, the method comprises administering a coagulation-effective amount of a CFXTENcomposition to the subject in need f In another embodiment, the method comprises the step ofadministering to the t with a bleed a ation-effective amount of a pharmaceutical compositionthat includes a CFXTEN, wherein the administration results in an arrest or attenuation of the bleeding.
As used herein, “coagulation-effective amount” is an amount of a FVIII composition that, whenadministered to a subject, is sufficient to effect hemostasis or other beneficial or d therapeutic(including preventative) result. In cing the present invention, it will be understood that acoagulation-effective amount can be administered in one or more administrations. Precise coagulationeffectiveamounts of the pharmaceutical composition to be stered will be guided by the judgmentof the practitioner, however, the unit dose will generally depend on the severity or cause of the bleedingand the amount of pre-eXisting FVIII in the subject. In a particular embodiment of the method of treatinga bleed, a ation-effective amount of a pharmaceutical compositions comprising CFXTEN isadministered to a subject ing from a bleeding episode, wherein the stration results in theresolution of the bleeding for a duration at least two-fold, or at least three-fold, or at least four-foldlonger compared to a FVIII not linked to XTEN and administered to a comparable subject with acomparable bleed at a comparable dose.
In r embodiment, the administration of a coagulation-effective amount of a CFXTENcomposition to a subject with a factor VIII-related condition results in a 10%, or 20%, or 30%, or 40%,or 50%, or 60%, or 70% or greater improvement of one or more biochemical, physiological or alparameters associated with the FVIII condition, compared to the FVIII not linked to XTEN, whenmeasured at between 2 and 7 days after administration. In another embodiment, the stration of acoagulation-effective amount of a CFXTEN composition to the subject in need thereof results in animprovement of one or more mical, physiological or clinical parameters associated with the FVIIIcondition for a period at least two-fold longer, or at least four-fold longer, or at least five-fold longer, orat least siX-fold longer ed to period achieved by a FVIII not linked to XTEN and administered at acomparable dose. Non-limiting examples of parameters that are improved for a longer duration includeblood concentrations of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, areduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a dchromogenic FVIII assay time, a reduced bleeding time, among other FVIII-related parameters known inthe art. In the foregoing embodiments of the paragraph, the administered CFXTEN comprises a FVIIIwith at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at leastabout 99% sequence identity to a factor VIII of Table l and one or more XTEN sequences with at leastabout 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99%sequence identity to an XTEN of Table 4 inserted into the FVIII at one or more ons selected fromTable 5, Table 6, Table 7, Table 8, and Table 9, or as depicted in FIGS. 8-9. In certain embodiments, atleast one XTEN insertion site of the CFXTEN is ed from amino acids 32, 220, 224, 336, 339, 390,399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human FVHI).
In a ular embodiment of the method of ent, a coagulation-effective amount ofCFXTEN fusion protein administered to a subject suffering from hemophilia A is sufficient to increasethe circulating FVIII gulant concentration to greater than 0.05 IU/ml and to maintain hemostasisfor at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at leastabout 120 h, or at least about 144 h, or at least about 168 h, or greater. In another embodiment, theadministration of a coagulation-effective amount of a pharmaceutical composition comprising CFXTENto a subject in need thereof results in a r reduction in a one-stage ng assay time of at leastabout 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, orabout 70%, or more in a blood sample from the subject at 2-7 days after the administration compared tothe assay time in a subject after administration of a comparable amount of the corresponding FVIII notlinked to XTEN. In another embodiment, the administration of a therapeutically effective amount of aCFXTEN or a pharmaceutical compositions comprising CFXTEN to a subject in need thereof s in agreater reduction in the activated partial prothrombin time of at least about 5%, or about 10%, or about%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a blood samplefrom the t 2-7 days after administration compared to the activated partial prothrombin time in asubject after administration of a comparable amount of the corresponding FVIII not linked to XTEN. Inanother embodiment, the administration of a CFXTEN or a pharmaceutical compositions comprisingCFXTEN to a subject in need thereof using a therapeutically effective amount results in nance ofactivated partial prothrombin times Within 30% of normal in a blood sample from the subject for a periodof time that is at least ld, or at least about fold, or at least about four-fold longer compared tothat of a FVIII not linked to XTEN and administered to a subject using a comparable dose.
In one ment of the method of treatment, the CFXTEN fusion protein is formulated andadministered as a ceutical composition comprising the CFXTEN in admixture With aceutically acceptable excipient. Methods for making pharmaceutical formulations are well knownin the art. Techniques and formulations lly may be found in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Co., , Pa. 1990 (See, also, Wang and Hanson, ParenteralFormulations of Proteins and Peptides: Stability and Stabilizers, Journal of Parenteral Science andTechnology Technical Report No., 10, Supp. 42-2S (1988)).
In another aspect, the invention provides a regimen for ng a hemophilia A patient, saidregimen comprising a composition comprising a CFXTEN fusion protein. In one embodiment of theregimen for treating a hemophilia A patient, the regimen further comprises the step of determining theamount of pharmaceutical composition comprising the CFXTEN needed to achieve hemostasis in thepatient. In some embodiments of the regimen, (i) a smaller 1U amount of about two-fold less, or aboutthree-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-foldless, or about 10-fold less of the pharmaceutical ition comprising CFXTEN is administered to asubject in need thereof in comparison to the corresponding coagulation factor not linked to the XTENunder an otherwise same dose regimen, and the fusion protein achieves a comparable area under thecurve (based on IU/ml) and/or a comparable therapeutic effect as the corresponding FVIII not linked tothe XTEN; (ii) the ceutical composition is administered less frequently (e. g., every three days,about every seven days, about every 10 days, about every 14 days, about every 21 days, or aboutmonthly) in comparison to the corresponding FVIII not linked to the XTEN under an otherwise samedose amount, and the fusion protein achieves a comparable area under the curve and/or a comparabletherapeutic effect as the ponding coagulation factor not linked to the XTEN; or (iii) anaccumulative smaller IU amount of at least about 20%, or about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90% less of the pharmaceutical composition isadministered in comparison to the corresponding FVIII not linked to the XTEN under an otherwise samedose schedule and the CFXTEN fusion protein es a comparable therapeutic effect as thecorresponding FVIII not linked to the XTEN. The accumulative smaller IU amount is measured for aperiod of at least about one week, or about 14 days, or about 21 days, or about one month. In theing embodiments, the therapeutic effect can be determined by any of the measured tersdescribed herein, including but not limited to blood tration of procoagulant FVIII, a reducedted partial prothrombin (aPTT) assay time, a d one-stage or two-stage ng assay time,delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a d bleeding time,resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII,fibrinogen levels, or other assays known in the art for assessing coagulopathies of FVIII. In anotherembodiment, the ion provides CFXTEN for use in a regimen for a treating a hemophilia A subjectcomprising administering an CFXTEN composition in two or more successive doses to the subject at aneffective amount, wherein the tration results in at least a 10%, or 20%, or 30%, or 40%, or 50%,or 60%, or 70%, or 80%, or 90% r improvement of at least one, two, or three parameters associatedwith the disease compared to a FVIII not linked to XTEN and administered using a comparable dose.
In one , the present invention relates to a method of preventing or treating the bleeding ina patient, optionally a haemophilia A patient, having pre-eXisting inhibitor(s) against FVIII. Inhibitoryantibodies against FVIII commonly develop in hemophiliacs, where the overall incidence of developingan inhibitor is 15 -3 0%, particularly in haemophiliacs who are heavily exposed to FVIII concentrates(Algiman et al. l dies to factor VIII (anti-hemophilic factor) in healthy individuals. PNASUSA (1992) 89: 3795-3799). However, inhibitory antibodies also occur in patients in auto-immunedisorders, malignancies (such as lymphoproliferative disorders, lymphomas and solid tumors), duringpregnancy and in the post-partum state. Inhibition can also occur when antibodies interfere with thebinding of FVIII to FIX and FX. Simultaneously or alternatively, anti-FVIII antibodies can interfere withthe binding of von Willebrand factor and/or phospholipids to FVIII, affecting coagulation and/or ifeof FVIII. The presence of inhibitory antibodies is often first detected with ms such as easybruising and uncontrolled bleeding, and is y referred to as acquired hemophilia. Anti-FVIIIantibodies can be determined by different methods including quantitation of anti-FVIII activity incoagulation assays, ELISA for FVIII inhibitors and purification using chromatography andimmunoadsorption (Algiman et al., 1992). Accordingly, the inventive methods are used in the treatmentor prevention of any condition associated with or characterized by the presence of inhibitory antibodiesto FVIII. In one embodiment, the invention provides a method of treating a patient haVing a pre-eXistinginhibitor t FVIII, the method comprising the step of administering to the patient a coagulation-effective amount of a CFXTEN fusion protein that must be administered to achieve hemostasis, nthe coagulation-effective amount of fusion protein administered is reduced in comparison to the amountof FVIII not linked to XTEN (or native FVIII) that must be stered to achieve hemostasis. In themethod, the reduced amount of CFXTEN is about two-fold, or three-fold, or four-fold, or five-fold less inIU/kg compared to the corresponding FVIII not linked to XTEN. In another embodiment of the method,the amount of CFXTEN that is administered as a dose to achieve hemostasis is at least 20 to 40 IU/kgless, or 30 to 60 IU/kg less, or 40 to 80 IU/kg less, or 60 to 100 IU/kg less, or 100 to 140 IU/kg less, or120 to 180 IU/kg less, or 140 to 200 IU/kg less compared to the corresponding FVIII not linked to XTENor to native FVIII ed to achieve hemostasis. In another embodiment, the invention provides amethod of treating a bleeding episode in a hemophilia A subject haVing a titer of at least 10, or 20, or 30,or 40, or 50, or 75, or 100, or 150, or 200 or more Bethesda units against a FVIII not linked to XTEN,wherein the dose of CFXTEN fusion protein required to arrest the ng epidose is at least two-fold,or three-fold, or four-fold, or five-fold, or six-fold, or fold, or eight-fold, or nine-fold, or 10-foldless in comparison to the amount of FVIII not linked to XTEN (or native FVIII) that must beadministered to achieve hemostasis in a comparable subject. It will be understood by one of skill in theart that the amount of gulant stered to in hemostasis will depend on the severity ofFVIII deficiency and/or the frequency or duration of bleeding.
A particular object of the present invention relates to use of CFXTEN with reduced g byFVIII tors that bind the A2 and/or C2 domains of Factor VIII as a drug. Such a drug isadvantageously used for maintaining hemostasis in a patient suffering from haemophilia, wherein suchpatient has ating FVIII inhibitors directed against the A2 domain and/or C2 domain of Factor VIII.
In one embodiment, the invention provides a method of treatment, the method comprising the step ofadministering to the patient with a A2 -binding inhibitor a coagulation-effective amount of aCFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%,or 60%, or 70%, or 80% or less binding to an inhibitor that binds the A2 domain of FVIII, ed tothe FVIII not linked to XTEN or to native FVIII, and wherein the stration results in hemostasis.
In another embodiment, the invention provides a method of ent, the method comprising the step ofadministering to the patient with a C2 domain-binding inhibitor a coagulation-effective amount of aCFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%,or 60%, or 70%, or 80% or less binding to an inhibitor that binds the C2 domain of FVIII, compared tothe FVIII not linked to XTEN or to native FVIII, and n the administration results in hemostasis.
The reduced binding of the subject CFXTEN can be assayed directly by ELISA that detects FVIIIinhibitors, or measured indirectly by demonstration of reduced inhibition of FVIII actiVity of theCFXTEN compared to native FVIII in the presence of an inhibitor as measured by a factor VIIIchromogenic test or one-step assay as described herein, or other suitable coagulation methods known inthe art. Alternatively, the subject CFXTEN can be measured for reduced (or absence of) tion in thepresence of known tors by use of a modified Bethesda assay. ing to a particular aspect ofthe present invention, a CFXTEN useful in the methods has reduced reactivity to one or more antibodiesfrom Table 10, as well as lly-occurring antibodies found in hemophilia ts. For gpurposes, such and other inhibitory antibodies can be obtained from humans (i.e. from the serum ofpatients which have inhibitory antibodies) or can be obtained from mice, guinea pigs, horses, goats, non-human primates and other mammals by immunization with FVIII, or fragments thereof, more ularlywith a nt comprising the all or part of the A2 or C2 domain, whether in polyclonal or monoclonalform.
The invention r contemplates that the CFXTEN used in accordance with the methodsprovided herein can be administered in conjunction with other treatment methods and itions (e. g.,other coagulation proteins) useful for treating factor VIII-related conditions, or conditions for whichcoagulation factor is adjunctive therapy; e. g., bleeding episodes due to injury or surgery.
] In another aspect, the invention provides methods of preparing a drug for a factor VIII-relatedcondition, comprising ing a factor VIII sequence selected from Table 1 with one or more XTENselected from Table 4 inserted in one or more insertion sites selected from Table 5, Table 6, Table 7,Table 8, and Table 9 to result in a drug that retains at least a portion of the activity of the native FVIII.
The invention provides a method of preparing a pharmaceutical composition, comprising the step ofcombining the drug of the ing embodiment with at least one pharmaceutically acceptable carrier.
In one embodiment of the method of preparing a drug for a factor VIII-related condition, the factor VIIIhas a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about97%, or at least about 99% sequence identity compared to a sequence selected from Table 1 and the oneor more XTEN has a sequence with at least about 80%, or at least about 90%, or at least about 95%, or atleast about 97%, or at least about 99% sequence identity compared to a ce selected from any oneof Tables 3, 4, and 13-17, or a fragment thereof, wherein the one or more XTEN are inserted in one ormore ons selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In a particular embodimentof the foregoing, at least one XTEN insertion site is selected from amino acids 32, 220, 224, 336, 339,390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human.In another embodiment of the method, the CFXTEN comprises a sequence with at least about80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequenceidentity compared to a sequence selected from any one of Table 21.
In another aspect, the invention provides a method of making the CFXTEN itions toachieve desired pharmacokinetic, cologic or pharmaceutical properties. In general, the steps inthe design and production of the inventive fusion protein compositions, as illustrated in FIGS. 11-13,include: (1) the selection of a FVIII (e. g., native proteins, sequences of Table 1, analogs or derivativeswith activity) to treat the particular condition; (2) selecting one or more XTEN (e. g., sequences with atleast 80% identity to sequences set forth in Table 4) that will confer the desired pharmacokinetic andphysicochemical characteristics on the ing CFXTEN (e.g., the administration of the CFXTENcomposition to a subject results in the fusion protein being maintained above 0.05-0.4 IU/ml for a greaterperiod ed to FVIII not linked to XTEN); (3) establishing a desired N— to C-terminus configurationof the CFXTEN to achieve the desired y or PK parameters (e.g., selecting one or more insertionsites from Table 5, Table 6, Table 7, Table 8, and Table 9); (4) establishing the design of the expressionvector encoding the configured CFXTEN; (5) transforming a suitable host with the sion vector;and (6) expressing and recovering the resultant isolated CFXTEN fusion protein. In one embodiment ofthe method of making CFXTEN, the XTEN for insertion are evaluated by the application of Equation IVto maximize the Ratio XTEN Radii for the fusion protein construct, with the XTEN resulting in valuesgreater than 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9. or 3.0 being preferred.
For those CFXTEN for which an se in half-life or an increased period of time spent above theminimum coagulation-effective concentration is desired, the XTEN chosen for incorporation generallyhave at least about 144, or about 288, or about 432, or about 576, or about 864, or about 875, or about912, or about 923 amino acid residues where a single XTEN is to be incorporated into the . Inanother embodiment, the CFXTEN comprises a first XTEN of the foregoing lengths, and at least asecond XTEN of about 36, or about 42, or about 72, or about 144, or about 288, or about 576, or about864, or about 875, or about 912, or about 923, or about 1000 or more amino acid residues. The onof the XTEN within the fusion protein can include one, two, three, four, five or more locations edfrom Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9. In one embodiment, the method ofdesign es an insertion ofXTEN into the FVIII of at least one site selected from amino acids 32,220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 red ve to maturenative human FVIII).
In another aspect, the invention provides s of making CFXTEN compositions toimprove ease of manufacture, result in increased stability, increased water solubility, and/or ease offormulation, as compared to the native FVIII. In one embodiment, the invention includes a method ofincreasing the water solubility of a FVIII comprising the step of linking the FVIII with at least about80%, or about 90%, or about 95% identity to a sequence from Table 1 to one or more XTEN at one, two,three, four, five or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS.8-9 wherein the XTEN is a ce with at least about 80%, or about 90%, or about 95% sequenceidentity compared to a sequence from any one of Tables 3, 4, and 13-17 such that a higher concentrationin soluble form of the resulting CFXTEN can be achieved, under physiologic conditions, compared to theFVIII in an un-fused state. In a particular embodiment, the CFXTEN comprises a FVIII linked to two,three, four, or five XTEN having at least about 24, or about 36, or about 48, or about 60, or about 72, orabout 84, or about 96, or about 144, or about 288 amino acid residues inserted at sites selected fromTable 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9, in which the solubility of the fusion proteinunder physiologic conditions is at least three-fold greater than the corresponding FVIII not linked toXTEN, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 60-fold orgreater than FVIII not linked to XTEN. Factors that contribute to the property ofXTEN to conferincreased water solubility of CFs when incorporated into a fusion protein e the high solubility ofthe XTEN fusion partner and the low degree of self-aggregation between molecules ofXTEN in solution,as well as expanding the hydrophilicity of FVIII al loops into which the XTEN is inserted. Insome ments, the method results in a CFXTEN fiJsion protein wherein the water solubility is atleast about 20%, or at least about 30% r, or at least about 50% greater, or at least about 75%greater, or at least about 90% greater, or at least about 100% greater, or at least about 150% greater, or atleast about 200% greater, or at least about 400% greater, or at least about 600% greater, or at least about800% greater, or at least about 1000% greater, or at least about 2000% greater under physiologicconditions, compared to the ed FVIII. In one embodiment, the XTEN of the CFXTEN fusionprotein is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared toa ce from any one of Tables 3, 4, and 13-17. In another embodiment, the invention includes amethod of increasing the shelf-life of a FVIII comprising the step of linking the FVIII with one or moreXTEN at one or more sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein theshelf-life of the resulting CFXTEN is extended compared to the FVIII in an un-fused state. As usedherein, life refers to the period of time over which the procoagulant activity of a FVIII or CFXTENthat is in solution, lyophilized or in some other storage formulation s stable without undue loss ofactivity or that remains within release specifications established for the pharmaceutical ition. AFVIII that degrades or aggregates generally has reduced functional activity or reduced bioavailabilitycompared to one that s in solution. Factors that contribute to the ability of the method to extendthe shelf life of FVIII when incorporated into a fusion protein include increased water solubility, reducedself-aggregation in solution, and increased heat ity of the XTEN fusion partner. In particular, thelow tendency ofXTEN to aggregate facilitates methods of formulating pharmaceutical preparationscontaining higher drug concentrations of CFs, and the heat-stability ofXTEN contributes to the propertyof CFXTEN fusion proteins to remain soluble and functionally active for extended periods. The methodresults in CFXTEN fusion proteins with prolonged or extended shelf-life that exhibit greater activityrelative to a FVIII standard that has been ted to the same storage and handling conditions. Therd may be the un—fused full-length FVIII or a cially-available FVIII pharmaceuticalition. In one embodiment, the method includes the step of formulating the isolated CFXTENwith one or more pharmaceutically acceptable excipients that e the ability of the XTEN to retainits unstructured conformation and for the CFXTEN to remain soluble in the formulation for a time that isgreater than that of the corresponding un—fused FVIII. In one embodiment, the method comprises linkinga FVIII selected from Table 1 to one or more XTEN selected from any one of Tables 3, 4, and 13-17inserted at one or more sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 and ngwith at least one pharmaceutically acceptable excipient to create a pharmaceutical ition thatretains greater than about 100% of the procoagulant activity, or greater than about 105%, 110%, 120%,130%, 150% or 200% of the procoagulant activity of a FVIII standard subjected to the same storage andhandling conditions when compared at a time point of at least 90 days, or at least 6 months, or at least 12WO 22617 months. Shelf-life may also be assessed in terms of functional activity ing after storage,normalized to functional activity when storage began. In some embodiments, CFXTEN pharmaceuticalcompositions of the invention retain about 50% more procoagulant activity, or about 60%, 70%, 80%, or90% more of the procoagulant ty of a FVIII standard when subjected to the same conditions for thesame period of up to 2 weeks, or 4 weeks, or 6 weeks or longer under various temperature conditions. Inone embodiment, the CFXTEN pharmaceutical composition retains at least about 50%, or about 60%, orat least about 70%, or at least about 80%, and most preferably at least about 90% or more of its originalactivity in solution when heated at 80°C for 10 min. In another embodiment, the CFXTENceutical composition retains at least about 50%, preferably at least about 60%, or at least about70%, or at least about 80%, or atively at least about 90% or more of its original activity in solutionwhen heated or maintained at 37°C for about 7 days. In another embodiment, CFXTEN pharmaceuticalcomposition retains at least about 80% or more of its onal activity after re to a temperatureof about 30°C to about 70°C over a period of time of about one hour to about 18 hours. In the foregoingembodiments above described in this paragraph, the retained activity of the CFXTENpharmaceutical compositions is at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold greater at a given time point than that of acorresponding ceutical composition comprising FVIII not linked to the XTEN.
VII). THE NUCLEIC ACIDS SEQUENCES OF THE INVENTION The present ion provides isolated polynucleic acids encoding CFXTEN chimeric fusionproteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimericfusion proteins, including homologous variants thereof. In another aspect, the invention encompassesmethods to produce polynucleic acids encoding CFXTEN chimeric fusion proteins and sequencescomplementary to polynucleic acid molecules encoding CFXTEN chimeric fusion protein, inghomologous variants thereof. In general, and as illustrated in FIGS. 11-13, the methods of producing acleotide sequence coding for a CFXTEN fusion protein and expressing the resulting gene productinclude assembling nucleotides encoding FVIII and XTEN, ligating the components in frame,orating the encoding gene into an sion vector appropriate for a host cell, transforming theappropriate host cell with the expression vector, and ing the host cell under conditions causing orpermitting the fusion protein to be expressed in the transformed host cell, thereby producing thebiologically-active CFXTEN polypeptide, which is recovered as an isolated fusion n by standardprotein purification methods known in the art. Standard recombinant techniques in molecular biology isused to make the cleotides and expression vectors of the present invention.
In accordance with the invention, nucleic acid sequences that encode CFXTEN (or itsment) are used to generate recombinant DNA molecules that direct the expression of CFXTENfusion proteins in appropriate host cells. For the purposes of the invention, nucleic acid encoding asignal peptide corresponding to that of native human FVIII ing MQIELSTCFFLCLLRFCFS (SEQID NO: 1611)) can be added to any of the encoding constructs described herein to aid in the expressionand secretion of the CFXTEN fusion protein. In one embodiment, the nucleic acid add isATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ IDNO: 1613), or the complement thereof.
Several cloning gies are suitable for performing the present invention, many of which isused to generate a construct that comprises a gene coding for a fusion protein of the CFXTENition of the present invention, or its complement. In some embodiments, the cloning strategy isused to create a gene that encodes a monomeric CFXTEN that comprises at least a first FVIII and at leasta first XTEN polypeptide, or their complement. In one embodiment of the foregoing, the gene sesa ce encoding a FVIII or sequence variant. In other embodiments, the cloning strategy is used tocreate a gene that encodes a monomeric CFXTEN that comprises nucleotides encoding at least a firstmolecule of FVIII or its complement and a first and at least a second XTEN or their complement that isused to transform a host cell for expression of the fusion protein of the CFXTEN composition. In theing embodiments hereinabove described in this paragraph, the genes can further comprisenucleotides ng spacer sequences that also encode cleavage sequence(s).
In designing a desired XTEN sequences, it was discovered that the non-repetitive nature of theXTEN of the inventive compositions is achieved despite use of a ”building block" molecular approach inthe on of the XTEN-encoding sequences. This was achieved by the use of a library ofpolynucleotides ng peptide sequence motifs, described above, that are then d and/ormultimerized to create the genes encoding the XTEN sequences (see FIGS. 11 and 12 and Examples).
Thus, while the ) of the expressed fusion protein may consist of multiple units of as few as fourent sequence motifs, because the motifs themselves consist of non-repetitive amino acid sequences,the overall XTEN sequence is rendered non-repetitive. Accordingly, in one embodiment, the XTEN-encoding polynucleotides comprise multiple polynucleotides that encode non-repetitive sequences, ormotifs, operably linked in frame and in which the resulting expressed XTEN amino acid sequences arenon-repetitive.
In one approach, a construct is first prepared containing the DNA sequence corresponding toCFXTEN fusion protein. DNA encoding the FVIII of the compositions is obtained synthetically, from acommercial source, or from a cDNA library ed using standard methods from tissue or isolated cellsbelieved to possess FVIII mRNA and to express it at a detectable level. If necessary, the codingsequence can be obtained using tional primer extension procedures as described in Sambrook, etal. to detect precursors and processing intermediates of mRNA that may not have been reverse-, supra,transcribed into cDNA. One can then use polymerase chain reaction (PCR) methodology to y thetarget DNA or RNA coding sequence to obtain sufficient material for the preparation of the CFXTENconstructs containing the FVIII gene. Assays can then be conducted to confirm that the hybridizing full-length genes are the d FVIII gene(s). By these conventional methods, DNA can be convenientlyobtained from a cDNA library prepared from such sources. The FVIII ng gene(s) can also createdby standard tic procedures known in the art (e. g., automated nucleic acid synthesis using, forexample one of the s described in Engels et al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), 2012/046326using DNA sequences obtained from ly available databases, patents, or literature references. Suchprocedures are well known in the art and well described in the scientific and patent literature. Forexample, sequences can be obtained from Chemical cts Services (CAS) Registry Numbers(published by the an Chemical Society) and/or GenBank Accession Numbers (e.g., Locus ID,NP_XXXXX, and XP_XXXXX) Model Protein identifiers available through the National Center forBiotechnology Information (NCBI) webpage, available on the world wide web at ncbi.nlm.nih.gov thatcorrespond to s in the CAS Registry or GenBank database that contain an amino acid sequence ofthe protein of st or of a fragment or variant of the protein. In one embodiment, the FVIII encodinggene encodes a protein sequence from Table l, or a fragment or variant thereof A gene or polynucleotide encoding the FVIII portion of the subject CFXTEN protein, in thecase of an expressed fusion protein that comprises a single FVIII, is then cloned into a construct, which isa d or other vector under control of appropriate transcription and translation sequences for highlevel protein expression in a biological system. In a later step, a second gene or polynucleotide codingfor the XTEN is genetically fused to the nucleotides encoding the N- and/or C-terminus of the FVIII geneby cloning it into the construct adjacent and in frame with the gene(s) coding for the FVIII. This secondstep occurs through a ligation or multimerization step. In the foregoing embodiments hereinabovedescribed in this paragraph, it is to be understood that the gene ucts that are created canalternatively be the complement of the respective genes that encode the respective fusion proteins.
The gene encoding for the XTEN can be made in one or more steps, either fully synthetically orby synthesis combined with tic processes, such as restriction enzyme-mediated cloning, PCR andoverlap extension, ing methods more fully described in the Examples. The methods disclosedherein can be used, for example, to ligate short sequences of polynucleotides encoding XTEN into longerXTEN genes of a desired length and sequence. In one embodiment, the method ligates two or morecodon—optimized oligonucleotides encoding XTEN motif or segment ces of about 9 to 14 aminoacids, or about 12 to 20 amino acids, or about 18 to 42 amino acids, or about 42 to about 144 aminoacids, or about 144 to about 288 amino acids, or 288 to about 864 amino acids or longer, or anyation of the foregoing ranges of motif or t lengths.
Alternatively, the disclosed method is used to multimerize XTEN-encoding sequences intolonger sequences of a desired length; e. g., a gene ng 36 amino acids ofXTEN can be dimerizedinto a gene encoding 72 amino acids, then 144, then 288, etc. Even with multimerization, XTENptides can be constructed such that the XTEN-encoding gene has low or virtually no repetitivenessh design of the codons selected for the motifs of the shortest unit being used, which can reducerecombination and increase stability of the encoding gene in the transformed host.
] Genes encoding XTEN with non-repetitive ces are assembled from oligonucleotidesusing standard techniques of gene synthesis. The gene design can be performed using algorithms thatoptimize codon usage and amino acid composition. In one method of the invention, a library ofrelatively short XTEN-encoding polynucleotide constructs is created and then assembled, as describedabove. The resulting genes are then assembled with genes ng FVIII or regions of FVIII, asrated in FIGS. 11 and 12, and the resulting genes used to transform a host cell and produce andrecover the CFXTEN for evaluation of its properties, as described herein.
In another aspect, the invention provides isolated nucleic acids comprising a polynucleotidesequence encoding the CFXTEN fusion protein embodiments described herein. In one embodiment, theisolated c acid comprises a polynucleotide sequence selected from (a) a ce having at leastabout 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identityed to a ce of comparable length selected from Table 21, when optimally aligned, or (b) thement of the polynucleotide of (a). In another embodiment, the isolated nucleic acid comprises thesequence ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT(SEQ ID NO: 1613) linked to the 5’ end of the nucleic acid of (a) or the complement of the sequencelinked to the 3’ end of (b). cleotide ies In another aspect, the invention provides libraries of polynucleotides that encode XTENsequences that are used to assemble genes that encode XTEN of a desired length and sequence.
In certain embodiments, the XTEN-encoding y constructs comprise polynucleotides thatencode polypeptide segments of a fixed length. As an initial step, a library of oligonucleotides thatencode motifs of 9-14 amino acid residues can be assembled. In a preferred embodiment, libraries ofoligonucleotides that encode motifs of 12 amino acids are assembled.
] The XTEN-encoding sequence segments can be dimerized or multimerized into longerencoding sequences, as depicted schematically in . Dimerization or erization can beperformed by ligation, overlap extension, PCR ly or similar cloning techniques known in the art.
This process of can be repeated multiple times until the resulting XTEN-encoding sequences havereached the organization of sequence and desired length, providing the XTEN-encoding genes. As willbe iated, a library of polynucleotides that encodes, e. g., 12 amino acid motifs can be zedand/or ligated into a library of polynucleotides that encode 36 amino acids. Libraries encoding motifs ofdifferent lengths; e. g., 9-14 amino acid motifs leading to libraries encoding 27 to 42 amino acids arecontemplated by the invention. In turn, the library of polynucleotides that encode 27 to 42 amino acids,and preferably 36 amino acids (as bed in the Examples) can be serially dimerized into a librarycontaining successively longer lengths of polynucleotides that encode XTEN sequences of a desiredlength for incorporation into the gene encoding the CFXTEN fusion protein, as disclosed herein.
A more efficient way to optimize the DNA sequence encoding XTEN is based oncombinatorial libraries. The gene encoding XTEN can be designed and synthesized in segment such thatmultiple codon versions are ed for each segment. These segments can be randomly assembled intoa library of genes such that each library member encodes the same amino acid sequences but librarys comprise a large number of codon versions. Such libraries can be screened for genes that resultin evel sion and/or a low abundance of truncation products. The process of combinatorialWO 22617 gene assembly is illustrated in . The genes in are assembled from 6 base fragments andeach fragment is available in 4 different codon versions. This allows for a theoretical diversity of 4096.
In some embodiments, libraries are assembled of polynucleotides that encode amino acids thatare limited to c sequence XTEN families; e. g., the AD, AE, AF, AG, AM, or AQ ces ofTable 4. In other embodiments, libraries comprise sequences that encode two or more of the motiffamily sequences from Table 3. The names and sequences of representative, non-limitingpolynucleotide sequences of libraries that encode 36mers are presented in Tables 13-17, and the methodsused to create them are bed more fully in the respective Examples. In other embodiments, librariesthat encode XTEN are constructed from segments of polynucleotide codons linked in a randomizedsequence that encode amino acids wherein at least about 80%, or at least about 90%, or at least about91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at leastabout 97%, or at least about 98%, or at least about 99% of the codons are selected from the groupconsisting of s for glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline(P) amino acids. The ies can be used, in turn, for serial dimerization or ligation to achievepolynucleotide sequence libraries that encode XTEN sequences, for example, of 42, 48, 72, 144, 288,576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well asintermediate lengths, in which the encoded XTEN can have one or more of the properties disclosedherein, when sed as a component of a CFXTEN filSiOIl protein. In some cases, the polynucleotidelibrary sequences may also include onal bases used as ”sequencing islands,” described more fullybelow. is a schematic flowchart of representative, non-limiting steps in the ly of aXTEN polynucleotide construct and a CFXTEN polynucleotide construct in the embodiments of theinvention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 aminoacid motif (“12-mer”), which is ligated to additional sequence motifs from a y to create a pool thatencompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of anoligo containing BbsI, and KpnI restriction sites 503. The resulting pool of ligation products is gelpurifiedand the band with the desired length ofXTEN is cut, resulting in an isolated XTEN gene with astopper sequence 505. The XTEN gene is cloned into a stuffer . In this case, the vector encodes anoptional CBD sequence 506 and a GFP gene 508. Digestion is than performed with BbsI/HindIII toremove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIIIed vector containing a gene encoding the FVIII, resulting in the gene 500 encoding an FVIII-XTEN fusion protein.
One may clone the library of XTEN-encoding genes into one or more expression sknown in the art. To facilitate the identification of xpressing library s, one can constructthe library as fusion to a reporter protein. Non-limiting examples of suitable reporter genes are greenfluorescent protein, luciferace, alkaline phosphatase, and beta-galactosidase. By screening, one canidentify short XTEN sequences that can be expressed in high concentration in the host organism ofchoice. Subsequently, one can generate a library of random XTEN dimers and repeat the screen for highlevel of expression. Subsequently, one can screen the resulting constructs for a number of propertiessuch as level of sion, protease stability, or binding to antiserum.
One aspect of the invention is to provide polynucleotide sequences ng the components ofthe fusion protein wherein the creation of the sequence has undergone codon optimization. Of particularinterest is codon optimization with the goal of improving expression of the polypeptide compositions andto improve the genetic stability of the encoding gene in the production hosts. For e, codonoptimization is of ular importance for XTEN ces that are rich in glycine or that have veryrepetitive amino acid sequences. Codon optimization is performed using computer programsfsson, C., et al. (2004) Trends hnol, 22: 346-53), some of which ze ribosomalpausing (Coda Genomics Inc.). In one embodiment, one can perform codon optimization by constructingcodon libraries where all members of the library encode the same amino acid sequence but where codonusage is varied. Such libraries can be screened for highly expressing and genetically stable members thatare particularly suitable for the large-scale production of XTEN-containing products. When ingXTEN sequences one can consider a number of properties. One can minimize the repetitiveness in theng DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely usedby the production host (e.g. the AGG and AGA arginine codons and one leucine codon in E. coli). In thecase of E. coli, two glycine codons, GGA and GGG, are rarely used in highly expressed proteins. Thuscodon optimization of the gene ng XTEN sequences can be very desirable. DNA ces thathave a high level of glycine tend to have a high GC content that can lead to instability or low expressionlevels. Thus, when possible, it is preferred to choose codons such that the GC-content of XTEN-encoding sequence is suitable for the production organism that will be used to manufacture the XTEN.
In one embodiment, polynucleotide libraries are constructed using the disclosed methodswherein all members of the library encode the same amino acid sequence but where codon usage for therespective amino acids in the sequence is varied or optimized for the intended host cell. Such librariescan be screened for highly sing and genetically stable members that are particularly suitable for thelarge-scale production of XTEN-containing products. In one embodiment, the libraries are zed forexpression in a eukaryotic host cell.
Optionally, one can sequence clones in the library to eliminate isolates that contain undesirablesequences. The initial library of short XTEN sequences allows some ion in amino acid ce.
For instance one can randomize some codons such that a number of hilic amino acids can occur ina particular position. During the process of iterative multimerization one can screen the resulting librarymembers for other characteristics like solubility or protease resistance in addition to a screen for high-level expression.
Once the gene that encodes the XTEN of d length and properties is selected, it isgenetically fused at the desired location to the nucleotides encoding the FVIII ) by cloning it intothe construct adjacent and in frame with the gene coding for FVIII, or alternatively between nucleotidesencoding adjacent s of the FVIII, or alternatively within a sequence encoding a given FVIIIdomain, or alternatively in frame with nucleotides encoding a spacer/cleavage sequence linked to aWO 22617 terminal XTEN. The ion provides various permutations of the foregoing, ing on theCFXTEN to be encoded. For example, a gene encoding a CFXTEN fusion protein sing a FVIIIand two XTEN, such as embodied by formula VI, as ed above, the gene would havepolynucleotides encoding FVIII, encoding two XTEN, which can be identical or different in compositionand sequence length. In one non-limiting embodiment of the foregoing, the FVIII polynucleotides wouldencode factor VIII and the polynucleotides encoding the C-terminus XTEN would encode an XTEN of288 amino acids and the polynucleotides encoding an internal XTEN adjacent to the C-terminus of theA2 domain would encode an XTEN of 144 amino acids. The step of cloning the FVIII genes into theXTEN construct can occur through a ligation or multimerization step, as shown in . Theconstructs encoding CFXTEN fusion ns can be designed in different configurations of thecomponents XTEN, CF, and spacer sequences, such as the configurations of formulae I-VIII. In oneembodiment, the construct comprises polynucleotide sequences complementary to, or those that encode amonomeric polypeptide of components in the ing order (5’ to 3’) FVIII, an XTEN internal to the Bdomain, and a C-terminal XTEN. In another ment, the construct comprises polynucleotidesequences complementary to, or those that encode a monomeric polypeptide of components in thefollowing order (5’ to 3’) FVIIII, spacer sequence linked to the C-terminus, and XTEN. The spacerpolynucleotides can optionally comprise sequences encoding cleavage ces. As will be apparent tothose of skill in the art, multiple permutations of FVIII domains and ed XTEN are possible.
Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences mayalso be determined conventionally by using known software or computer ms such as the BestFit orGap pairwise ison programs (GCG Wisconsin Package, Genetics Computer Group, 575 ScienceDrive, Madison, Wis. 53711). t uses the local homology algorithm of Smith and Waterman(Advances in Applied Mathematics. 1981. 2: 482-489), to find the best segment of identity or similaritybetween two sequences. Gap performs global alignments: all of one sequence with all of r similarsequence using the method leman and Wunsch, (Journal of Molecular y. 1970. 48:443-453). When using a sequence alignment program such as BestFit, to determine the degree of sequencehomology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may beselected to optimize identity, similarity or homology scores.
Nucleic acid sequences that are “complementary” are those that are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences that are substantially complementary, as maybe assessed by the same nucleotide ison set forth above, or as defined as being capable ofhybridizing to the polynucleotides that encode the CFXTEN sequences under stringent conditions, suchas those described herein.
The ing polynucleotides encoding the CFXTEN chimeric fusion proteins can then beindividually cloned into an expression vector. The nucleic acid sequence is ed into the vector by avariety of procedures. In l, DNA is inserted into an riate restriction endonuclease site(s)using techniques known in the art. Vector components generally include, but are not limited to, one ormore of a signal sequence, an origin of ation, one or more marker genes, an er element, apromoter, and a transcription termination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation ques which are known to the skilled n.
Such techniques are well known in the art and well described in the ific and patent literature.
Various vectors are publicly available. The vector may, for example, be in the form of aplasmid, cosmid, Viral particle, or phage that may conveniently be subjected to recombinant DNAprocedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as anextrachromosomal entity, the replication of which is independent of chromosomal ation, e. g., aplasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated intothe host cell genome and replicated together with the chromosome(s) into which it has been integrated.
Representative plasmids are illustrated in , with encoding regions for ent configurations ofFVIII and XTEN components portrayed.
The invention provides for the use of plasmid vectors containing ation and controlsequences that are compatible with and recognized by the host cell, and are ly linked to theCFXTEN gene for controlled expression of the CFXTEN fusion proteins. The vector ordinarily carries areplication site, as well as sequences that encode proteins that are capable of providing phenotypicion in transformed cells. Such vector sequences are well known for a variety of bacteria, yeast, andViruses. Useful expression vectors that can be used e, for example, segments of chromosomal,non-chromosomal and synthetic DNA sequences. ”Expression vector” refers to a DNA constructcontaining a DNA sequence that is operably linked to a suitable control sequence capable of effecting theexpression of the DNA encoding the fusion protein in a le host. The requirements are that thevectors are replicable and Viable in the host cell of choice. Low— or high-copy number vectors may beused as desired.
Other suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA andknown bacterial ds such as col El, pCRl, pBR322, 2, pET, pGEX as described by Smith, etal., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such asthe numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives ofthe 2m plasmid, as well as centomeric and ative yeast shuttle vectors; vectors useful in eukaryoticcells such as vectors useful in insect or ian cells; s derived from combinations of plasmidsand phage DNAs, such as plasmids that have been modified to employ phage DNA or the expressioncontrol sequences; and the like. Yeast expression systems that can also be used in the present inventioninclude, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion sA, B, C(Invitrogen), pRS vectors and the like.
The control sequences of the vector include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encoding suitable mRNA ribosome gsites, and ces that control termination of transcription and translation. The promoter may be anyWO 22617 DNA sequence, Which shows transcriptional activity in the host cell of choice and may be derived fromgenes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the ription of the DNA encoding the FVIHpolypeptide variant in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1(1981), 854-864), the MT-l (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell 41 :521-530, 1985) or the adenovirus 2 major late promoter(Kaufman and Sharp, Mol. Cell. Biol, -1319, 1982). The vector may also carry sequences such asUCOE (ubiquitous chromatin opening elements).
Examples of suitable promoters for use in filamentous fungus host cells are, for instance, theADH3 promoter or the tpiA promoter. Examples of other useful promoters are those derived from thegene ng A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger l (X-amylase, A. niger acid stable (x-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor mieheilipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
Preferred are the TAKA—amylase and gluA promoters.
Promoters suitable for use in expression vectors with prokaryotic hosts include the B-lactamaseand lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281 :544(1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057(1980); EP 36,776], and hybrid ers such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.
USA, 80:21-25 (1983)], all is operably linked to the DNA encoding CFXTEN polypeptides. Promotersfor use in bacterial systems can also contain a Shine-Dalgarno (S.D.) sequence, ly linked to theDNA encoding CFXTEN polypeptides.
The invention plates use of other expression systems including, for example, abaculovirus expression system With both non-fusion transfer vectors, such as, but not limited to pVL941s, et al., Virology -402 ), pVL1393 (Invitrogen), pVL1392 (Summers, et al.,Virology 84:390- 402 (1978) and anitrogen) and pBlueBaclll (anitrogen), and fusion transfer vectorssuch as, but not d to, pAc7 00 (Summers, et al., Virology 84:390-402 (1978)), pAc701 and pAc70-2 (same as pAc700, With different reading frames), pAc36O anitrogen) and pBlueBacHisA, B, C (;Invitrogen) can be used.
Examples of suitable promoters for directing the transcription of the DNA encoding the FVIHpolypeptide t in mammalian cells are the CMV promoter (Boshart et al., Cell 41 :521-530, 1985),the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., Science 222 (1983), 809-814), the adenovirus 2 major late er(Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences such asUCOE (ubiquitous chromatin opening elements).
The DNA ces encoding the CFXTEN may also, if ary, be operably connected to asuitable terminator, such as the hGH terminator ter et al., Science 222, 1983, pp. 809-814) or theTP11 terminators (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 4) or ADH3 (McKnight etal., The EMBO J. 4, 1985, pp. 2093-2099). Expression vectors may also contain a set of RNA splice siteslocated ream from the promoter and upstream from the insertion site for the CFXTEN sequenceitself, including splice sites obtained from adenovirus. Also contained in the expression s is apolyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylationsignals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the adenovirus 5 Elb region, the hGH terminator (DeNoto et al. Nucl. AcidsRes. 93719-3730, 1981). The expression vectors may also include a noncoding viral leader sequence,such as the adenovirus 2 tripartite leader, d between the promoter and the RNA splice sites; andenhancer sequences, such as the SV40 enhancer.
To direct the CFXTEN of the present invention into the secretory pathway of the host cells, asecretory signal sequence (aka, a leader sequence, a prepro sequence, or a pre sequence) may beincluded in the recombinant vector. The secretory signal ce is operably linked to the DNAsequences encoding the CFXTEN, usually positioned 5’ to the DNA sequence encoding the CFXTENfusion protein. The secretory signal sequence may be that, normally associated with the native FVIIIprotein or may be from a gene encoding another secreted protein. Non-limiting examples include OmpA,PhoA, and DsbA for E. coli expression, ppL-alpha, DEX4, invertase signal peptide, acid phosphatasesignal peptide, CPY, or INUl for yeast expression, and 1L2L, SV40, IgG kappa and IgG lambda formammalian expression. Signal sequences are lly proteolytically d from the protein duringthe translocation and secretion process, generating a defined N-terminus. Methods are disclosed inArnau, er al., Protein sion and Purification 48: 1-13 (2006).
The procedures used to ligate the DNA sequences coding for the , the promoter andoptionally the terminator and/or ory signal sequence, respectively, and to insert them into levectors ning the information necessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook, J. et al., “Molecular g: A Laboratory Manual,” 3ml n, ColdSpring Harbor Laboratory Press, 2001). In this manner, a chimeric DNA molecule coding for aric CFXTEN fusion protein is generated within the construct. Optionally, this chimeric DNAmolecule may be transferred or cloned into another construct that is a more appropriate expressionvector. At this point, a host cell capable of expressing the chimeric DNA le can be transformedwith the chimeric DNA molecule.
Non-limiting examples of mammalian cell lines for use in the present ion are the COS-1(ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), BHK-570 cells (ATCC CRL 10314), CHO-Kl(ATCC CCL 61), CHO-S (Invitrogen 11619-012), and 293-F (Invitrogen R790-7), and the parental andderivative cell lines known in the art useful for expression of FVIII. A tk-ts13 BHK cell line is alsoavailable from the ATCC under accession number CRL 1632. In addition, a number of other cell linesmay be used within the present invention, including Rat Hep I (Rat ma; ATCC CRL 1600), RatHep 11 (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065),NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells b and Chasin, Proc. Natl.
Acad. Sci. USA 77:4216-4220, 1980).
Examples of suitable yeasts cells include cells of Saccharomyces spp. or Schizosaccharomycesspp., in particular strains of Saccharomyces siae or romyces kluyveri . Methods fortransforming yeast cells With heterologous DNA and producing heterologous ptides there from aredescribed, e.g. in US. Pat. No. 4,599,311, US. Pat. No. 4,931,373, US. Pat. No. 4,870,008, 5,037,743,and US. Pat. No. 4,845,075, all of Which are hereby incorporated by reference. Transformed cells areselected by a phenotype determined by a selectable marker, commonly drug ance or the ability togrow in the absence of a particular nutrient, e. g. leucine. A preferred vector for use in yeast is the PCT]vector disclosed in US. Pat. No. 4,931,373. The DNA sequences encoding the CFXTEN may beed by a signal sequence and optionally a leader sequence, e.g. as described above. Furtherexamples of suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula e. g. H.polymorpha or Pichia P. is (cf. Gleeson et al., J. Gen. Microbiol., , e.g. 132, 1986, pp. 3459-3465;US. Pat. No. 279). Examples of other fungal cells are cells of filamentous fungi, e. g. Aspergillusspp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains ofA. oryzae, A. nidulansor A. niger. The use ofAspergillus spp. for the expression of proteins is described in, e. g., EP 272 277,EP 238 023, EP 184 438 The ormation of F. oxysporum may, for instance, be carried out asdescribed by ier et al., 1989 Gene 78: 147-156. The transformation of Trichoderma spp. may beperformed for instance as described in EP 244 234.
Other suitable cells that can be used in the t invention include, but are not limited to,prokaryotic host cells strains such as Escherichia coli, (e. g., strain DH5-oc), Bacillus subtilis, Salmonellatyphimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus. Non-limitingexamples of suitable prokaryotes include those from the genera: Actinoplanes; Archaeoglobus;Bdellovibrio; Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus; ia;Oceanobacillus; ccus; Pseudomonas; Staphylococcus; Streptococcus; Streptomyces;Thermoplasma; and Vibrio.
Methods of transfecting mammalian cells and expressing DNA sequences introduced in thecells are described in e. g., Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.
Mol. Appl. Genet. 1 , 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426;Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Grahamand van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 5.
Cloned DNA sequences are introduced into ed mammalian cells by, for example, calciumphosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, SomaticCell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973), ection Withmany commercially available reagents such as FuGENEG Roche Diagnostics, Mannheim, Germany) orlipofectamine ogen) or by electroporation (Neumann et al., EMBO J. 1:841-845, 1982). Toidentify and select cells that express the exogenous DNA, a gene that confers a selectable phenotype (aable marker) is generally introduced into cells along With the gene or cDNA of interest. Preferredselectable markers include genes that confer resistance to drugs such as neomycin, hygromycin,cin, zeocin, and methotrexate. The selectable marker may be an amplifiable selectable marker. Apreferred amplif1able selectable marker is a dihydrofolate reductase (DHFR) ce. Further examplesof selectable markers are well known to one of skill in the art and include reporters such as enhancedgreen fluorescent protein (EGFP), beta-galactosidase (B-gal) or chloramphenicol transferase(CAT). able markers are reviewed by Thilly (Mammalian Cell logy, ButterworthPublishers, Stoneham, Mass., incorporated herein by reference). The person skilled in the art will easilybe able to choose suitable able markers. Any known selectable marker may be employed so long asit is e of being expressed simultaneously with the c acid encoding a gene product.
Selectable markers may be uced into the cell on a separate plasmid at the same time asthe gene of interest, or they may be introduced on the same plasmid. If, on the same plasmid, theselectable marker and the gene of interest may be under the control of different promoters or the samepromoter, the latter ement producing a dicistronic message. Constructs of this type are known inthe art (for example, Levinson and Simonsen, US. Pat. No. 4,713,339). It may also be advantageous toadd additional DNA, known as “carrier DNA,” to the mixture that is introduced into the cells.
After the cells have taken up the DNA, they are grown in an riate grth medium,typically 1-2 days, to begin expressing the gene of interest. As used herein the term “appropriate grthmedium” means a medium containing nts and other components required for the grth of cellsand the expression of the CFXTEN of interest. Media generally e a carbon , a nitrogensource, essential amino acids, essential sugars, Vitamins, salts, phospholipids, protein and grth factors.
For production of gamma-carboxylated proteins, the medium will contain Vitamin K, preferably at aconcentration of about 0.1 [Lg/ml to about 5 [Lg/ml. Drug selection is then d to select for the grthof cells that are expressing the selectable marker in a stable n. For cells that have been transfectedwith an amplif1able selectable marker the drug concentration may be increased to select for an increasedcopy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfectedcells are then screened for expression of the FVIH polypeptide variant of interest.
The transformed or transfected host cell is then cultured in a suitable nutrient medium underconditions permitting expression of the CFXTEN polypeptide after which the resulting peptide may bered from the culture as an isolated fusion protein. The medium used to culture the cells may be anyconventional medium suitable for growing the host cells, such as minimal or complex media containingriate supplements. le media are available from commercial suppliers or may be preparedaccording to published recipes (e. g. in catalogues of the American Type Culture Collection). The cultureconditions, such as temperature, pH and the like, are those usly used with the host cell selected forexpression, and will be apparent to the ordinarily skilled n.
Gene expression may be measured in a sample directly, for example, by conventional Southernblotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA,77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriatelylabeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed thatcan recognize specif1c duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carriedout where the duplex is bound to a surface, so that upon the formation of duplex on the surface, thepresence of dy bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological of fluorescent methods,such as immunohistochemical staining of cells or tissue sections and assay of cell e or body fluidsor the detection of selectable markers, to quantitate directly the expression of gene product. Antibodiesuseful for histochemical staining and/or assay of sample fluids may be either onal orpolyclonal, and may be prepared in any mammal. iently, the dies may be prepared against anative sequence FVIII polypeptide or against a synthetic e based on the DNA sequences providedherein or against exogenous sequence fused to FVIII and encoding a specific antibody epitope.
Examples of selectable markers are well known to one of skill in the art and include reporters such asenhanced green fluorescent n (EGFP), beta-galactosidase (5-gal) or chloramphenicolacetyltransferase (CAT).
Expressed CFXTEN polypeptide product(s) may be purified via methods known in the art orby methods disclosed herein. Procedures such as gel ion, affinity purification (e. g., using an anti-FVIII antibody column), salt fractionation, ion exchange chromatography, size exclusionchromatography, yapatite adsorption chromatography, hydrophobic interaction chromatographyand gel electrophoresis may be used; each tailored to r and purify the fusion protein produced bythe respective host cells. Additional purification may be achieved by conventional chemical purificationmeans, such as high performance liquid chromatography. Some expressed CFXTEN may requirerefolding during isolation and purification. Methods of purification are described in Robert K. Scopes,Protein Purification: Principles and Practice, Charles R. Castor (ed.), Springer-Verlag 1994, andSambrook, et al., supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev.
Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994). For therapeuticpurposes it is red that the CFXTEN fusion proteins of the invention are substantially pure. Thus, ina preferred embodiment of the ion the CFXTEN of the invention is purified to at least about 90 to95% neity, preferably to at least about 98% homogeneity. Purity may be assessed by, e. g., gelophoresis, HPLC, and amino-terminal amino acid cing..
VIII). PHARMACEUTICAL COMPOSITIONS The present invention provides pharmaceutical compositions comprising CFXTEN. In oneembodiment, the pharmaceutical composition comprises a CFXTEN fusion n disclosed hereinadmixed with at least one pharmaceutically acceptable carrier. CFXTEN polypeptides of the presention can be formulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the polypeptide is combined in admixture with a pharmaceutically acceptablecarrier vehicle, such as aqueous solutions, buffers, solvents and/or pharmaceutically acceptablesuspensions, emulsions, stabilizers or excipients. Examples of non-aqueous solvents include propylethylene glycol, polyethylene glycol and vegetable oils. Formulations of the pharmaceuticalcompositions are prepared for storage by mixing the active CFXTEN ingredient having the desireddegree of purity with optional physiologically acceptable carriers, excipients (e. g., sodium chloride, acalcium salt, sucrose, or polysorbate) or stabilizers (e. g., e, trehalose, se, arginine, a calciumsalt, glycine or histidine), as described in ton's Pharmaceutical Sciences 16th n, Osol, A.
Ed. (1980), in the form of lyophilized formulations or aqueous solutions.
The pharmaceutical composition may be supplied as a lyophilized powder to be reconstitutedprior to administration. In another embodiment, the pharmaceutical composition may be supplied in aliquid form in a Vial, the contents of which can be administered directly to a patient. Alternatively, thecomposition is supplied as a liquid in a pre-filled syringe for administration of the composition. Inanother embodiment, the composition is supplied as a liquid in a pre-fllled Vial that can be incorporatedinto a pump.
The pharmaceutical itions can be administered by any suitable means or route,including subcutaneously, subcutaneously by on pump, intramuscularly, and intravenously. It willbe appreciated that the preferred route will vary with the e and age of the recipient, and the severityof the ion being treated.
In one embodiment, the CFXTEN pharmaceutical composition in liquid form or afterreconstitution (when supplied as a lyophilized ) comprises coagulation factor VIII with an actiVityof at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least 300 IU/ml, or at least 400IU/ml, or an actiVity of at least 500 IU/ml, or an actiVity of at least 600 IU/ml, which composition iscapable of increasing factor VIII actiVity to at least 1.5% of the normal plasma level in the blood for atleast about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or atleast about 96 hours, or at least about 120 hours after stration of the factor VIII pharmaceuticalcomposition to a subject in need of e prophylaxis. In another embodiment, the CFXTENpharmaceutical composition in liquid form or after reconstitution (when supplied as a lyophilizedpowder) comprises coagulation factor VIII with an actiVity of at least 50 IU/ml, or at least 100 IU/ml, orat least 200 IU/ml, or at least 300 IU/ml, or at least 400 IU/ml, or at least 500 IU/ml, or an actiVity of atleast 600 IU/ml, which composition is capable of increasing factor VIII actiVity to at least 2.5% of thenormal plasma level in the blood for at least about 12 hours, or at least about 24 hours, or at least about48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours afteradministration to a subject in need of routine laxis. It is specifically plated that thepharmaceutical compositions of the foregoing can be formulated to include one or more ents,buffers or other ingredients known in the art to be compatible with administration by the intravenousroute or the subcutaneous route or the intramuscular route. Thus, in the embodiments hereinabovedescribed in this paragraph, the pharmaceutical composition is administered subcutaneously,intramuscularly or intravenously.
The compositions of the invention may be formulated using a variety of excipients. Suitableexcipients include microcrystalline ose (e. g. Avicel PH102, Avicel PH101), polymethacrylate,thyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (such asEudragit RS-3OD), hydroxypropyl methylcellulose (Methocel K1 00M, m CR Methocel K1 00M,el E5, Opadry®), ium stearate, talc, triethyl citrate, aqueous ethylcellulose dispersion(Surelease®), and protamine sulfate. The slow release agent may also comprise a carrier, which cancomprise, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicand absorption delaying agents. Pharmaceutically acceptable salts can also be used in these slow releaseagents, for e, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as wellas the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The compositionmay also n liquids, such as water, saline, glycerol, and ethanol, as well as substances such aswetting agents, emulsifying agents, or pH buffering . Liposomes may also be used as a carrier.
In another embodiment, the compositions of the present invention are ulated inliposomes, which have demonstrated utility in delivering beneficial active agents in a controlled mannerover prolonged periods of time. Liposomes are closed bilayer membranes containing an entrappedaqueous volume. Liposomes may also be unilamellar es sing a single membrane bilayer ormultilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueouslayer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails ofthe lipid are oriented toward the center of the bilayer while the hilic (polar) heads orient towardsthe aqueous phase. In one embodiment, the liposome may be coated with a flexible water solublepolymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver andspleen. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG,polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate,hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilicpeptide ces as described in US. Pat. Nos. 024; 6,126,966; 6,056,973; 6,043,094, thecontents of which are incorporated by reference in their entirety. Additional liposomal technologies aredescribed in US. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 191; 6,126,966;6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 4,684,479, the contents ofwhichare incorporated herein by reference. These be mes and lipid-coated microbubbles, andmethods for their manufacture. Thus, one skilled in the art, considering both the disclosure of thision and the sures of these other patents could produce a liposome for the extended release ofthe polypeptides of the present ion.
For liquid formulations, a desired property is that the formulation be supplied in a form that canpass h a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, orsubcutaneous administration.
Syringe pumps may also be used as slow release agents. Such devices are described in US.
Pat. Nos. 4,976,696; 185; 5,017,378; 6,309,370; 6,254,573; 173; 4,398,908; 6,572,585;,298,022; 5,176,502; 5,492,534; 5,318,540; and 4,988,337, the contents of which are incorporatedherein by reference. One skilled in the art, ering both the disclosure of this invention and thedisclosures of these other patents could produce a syringe pump for the extended release of thecompositions of the present ion.
IX). PHARMACEUTICAL KITS In another aspect, the invention provides a kit to facilitate the use of the CFXTENpolypeptides. The kit comprises the pharmaceutical composition provided herein, a container and a labelor package insert on or associated with the container. Suitable itiers include, for example, bottles,Vials, syringes, etc, formed frem a variety 0f materials such as glass or c. The container holds apharmaceutical composition as a fermulatien that is effective for treating the elated ion andmay have a sterile access pert (for example the ner maybe an intravenous on bag or a vialhaving a stepper pierceable by a hypedermie injection needle). The package insert can list the approvedindications for the drug, instructions for the reconstitution and/or stration of the drug for the usefor the approved indication, appropriate dosage and safety information, and information identifying thelot and expiration of the drug. In another embodiment of the foregoing, the kit can comprise a secondcontainer that can carry a suitable diluent for the pharmaceutical composition, the use of which willprovide the user with the appropriate tration to be delivered to the subject.
Example 1: uction ofXTEN_AD36 motif segments The following example describes the construction of a collection of codon—optimized genesencoding motif sequences of 36 amino acids. As a first step, a stuffer vector pCWO359 was constructedbased on a pET vector and that includes a T7 promoter. pCWO359 encodes a cellulose binding domain(CBD) and a TEV protease recognition site followed by a stuffer sequence that is flanked by BsaI, BbsI,and KpnI sites. The BsaI and BbsI sites were inserted such that they generate compatible overhangs afterdigestion. The stuffer sequence is followed by a truncated version of the GFP gene and a His tag. Thestuffer sequence contains stop codons and thus E. coli cells carrying the stuffer plasmid pCWO359 formnon-fluorescent es. The stuffer vector pCWO359 was digested with BsaI and KpnI to remove thestuffer segment and the resulting vector fragment was isolated by agarose gel purification. Thesequences were designated XTEN_AD3 6, reflecting the AD family of motifs. Its segments have theamino acid sequence [X]3 where X is a 12mer peptide with the sequences: GESPGGSSGSES (SEQ IDNO: 19), GPGESS (SEQ ID NO: 20), GSSESGSSEGGP (SEQ ID NO: 21), orGSGGEPSESGSS (SEQ ID NO: 22). The insert was obtained by annealing the following pairs ofphosphorylated tic oligonucleotide pairs:Aleor: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC (SEQ ID NO: 1619)ADlrev: YTCRGAACCGCTRGARCCACCHGGAGATTC (SEQ ID NO: 1620)AD2for: AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC (SEQ ID NO: 1621)AD2rev: ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT (SEQ ID NO: 1622)AD3for: AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC (SEQ ID NO: 1623) PCT/U82012/046326AD3reV: ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA (SEQ ID NO: 1624)AD4for: AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC (SEQ ID NO: 1625) We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylatedoligonucleotide pr_3KpnIstopperReV: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealedoligonucleotide pairs were ligated, which resulted in a mixture of products with varying length thatrepresents the varying number of 12mer repeats ligated to one pnI segment. The productscorresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gelelectrophoresis and ligated into the BsaI/KpnI ed r vector pCWO35 9. Most of the clones inthe resulting library ated LCWO401 showed green fluorescence after induction, which shows thatthe sequence ofXTEN_AD36 had been ligated in frame with the GFP gene and that most sequences ofXTEN_AD36 had good expression .
We screened 96 isolates from library LCWO401 for high level of fluorescence by stamping themonto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates wereidentified that contained segments with 36 amino acids as well as strong fluorescence. These eswere sequenced and 39 clones were identified that contained correct XTEN_AD36 segments. The filenames of the nucleotide and amino acid ucts and the sequences for these segments are listed inTable 13.
Table 13: DNA and Amino Acid Seguences for AD 36-mer motifs (SEQ ID NOS 203-278,res ectivel in order of a earanceFile name Amino acid sequence Nucleotide sequenceLCWO401_001_ GSGGEPSESGSSGESPGG GGTGGCGAACCGTCCGAGTCTGGTAGCTCAGFP-\_A0 1 .ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGCTCTAGCGGTTCCGAGTCAGGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCALCWO401_002_ GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCAGFP-\_B01 .ab1 SSGSESGSSESGSSEGGP GGTGAATCTCCTGGTGGTTCCAGCGGTTCTGAATCAGGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCALCWO401_003_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGGTCCAGFP-\_C0 1 .ab1 SSEGGPGESPGGSSGSES GGTTCCTCTGAAAGCGGTTCTTCTGAGGGTGGTCCAGGTGAATCTCCGGGTGGCTCCAGCGGTTCCGAGTCALCWO401_004_ SESGSSGSSESG GGTTCCGGTGGCGAACCGTCTGAATCTGGTAGCTCAGFP-\_D0 1 .abl SSEGGPGSGGEPSESGSS GGTTCTTCTGAAAGCGGTTCTTCCGAGGGTGGTCCAGGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCALCWO401_007_ GSSESGSSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTGGTCCAGFP-\_F0 1 .ab1 GSEGSSGPGESS GGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCALCWO401_008_ GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCAGFP-\_G0 1 .ab1 GSEGSSGPGESS GGTGAATCTCCAGGTGGTTCCAGCGGTTCTGAGTCAGGTAGCGAAGGTTCTTCTGGTCCAGGTGAATCCTCALCWO401_012_ GSGGEPSESGSSGSGGEP GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAGCTCAH0 1 .ab1 SESGSSGSEGSSGPGESS GGTTCCGGTGGCGAACCATCCGAATCTGGTAGCTCAGGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCALCWO401_015_ GSSESGSSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCAGFP-\_A02.ab1 GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA1_016_ GSSESGSSEGGPGSSESG GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCAGFP-\_B02.ab1 SSEGGPGSSESGSSEGGP GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCAFile name Amino acid sequence tide sequenceLCW0401_020_ GSGGEPSESGSSGSEGSS GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCAGFP-\_E02.ab1 GPGESSGSSESGSSEGGP GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCAGGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCALCW0401_022_ GSGGEPSESGSSGSSESG GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCAGFP-\_F02.ab1 SSEGGPGSGGEPSESGSS GGTTCTTCCGAAAGCGGTTCTTCTGAAGGTGGTCCAGGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCALCW0401_024_ SESGSSGSSESG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCAGFP-\_G02.ab1 GESPGGSSGSES GGTTCCTCCGAAAGCGGTTCTTCTGAAGGTGGTCCAGGTGAATCTCCAGGTGGTTCTAGCGGTTCTGAATCALCW0401_026_ SESGSSGESPGG GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAGCTCAGFP-\_H02.ab1 SSGSESGSEGSSGPGESS GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAATCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCALCW0401_027_ GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCAGFP-\_A03.ab1 SSGSESGSGGEPSESGSS GGTGAATCTCCGGGTGGTTCTAGCGGTTCTGAGTCAGGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCALCW0401_028_ GSSESGSSEGGPGSSESG TCTGAAAGCGGTTCTTCTGAGGGCGGTCCAGFP-\_B03.ab1 GSSESGSSEGGP TCCGAAAGCGGTTCTTCCGAGGGCGGTCCAGGTTCTTCCGAAAGCGGTTCTTCTGAAGGCGGTCCALCW0401_030_ GESPGGSSGSESGSEGSS GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAGTCAGFP-\_C03.ab1 GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCCGGTCCGGGTGAGTCCTCAGGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCALCW0401_031_ GSGGEPSESGSSGSGGEP GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCAGFP-\_D03.ab1 SESGSSGSSESGSSEGGP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCAGGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCALCW0401_033_ GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCAGFP-\_E03.ab1 SESGSSGSGGEPSESGSS GGTTCCGGTGGCGAACCATCCGAGTCTGGTAGCTCAGGTTCCGGTGGTGAACCATCCGAGTCTGGTAGCTCALCW0401_037_ GSGGEPSESGSSGSSESG GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCAGFP-\_F03.ab1 SSEGGPGSEGSSGPGESS GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCAGGTAGCGAAGGTTCTTCTGGTCCGGGCGAGTCTTCALCW0401_038_ GSGGEPSESGSSGSEGSS GGTTCCGGTGGTGAACCGTCCGAGTCTGGTAGCTCAGFP-\_G03.ab1 GPGESSGSGGEPSESGSS GAAGGTTCTTCTGGTCCGGGTGAGTCTTCAGGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCALCW0401_039_ SESGSSGESPGG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCAGFP-\_H03.ab1 SSGSESGSGGEPSESGSS GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCAGGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCALCW0401_040_ GSSESGSSEGGPGSGGEP GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCAGFP-\_A04.ab1 SESGSSGSSESGSSEGGP GGTTCCGGTGGTGAACCATCTGAATCTGGTAGCTCAGGTTCTTCTGAAAGCGGTTCTTCTGAAGGTGGTCCALCW0401_042_ GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCAGFP-\_C04.ab1 SSGSESGSEGSSGPGESS GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCAGGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCCTCALCW0401_046_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGGTCCAGFP-\_D04.ab1 SSEGGPGSSESGSSEGGP TCCGAAAGCGGTTCTTCTGAGGGCGGTCCAGGTTCCTCCGAAAGCGGTTCTTCTGAGGGTGGTCCALCW0401_047_ GSGGEPSESGSSGESPGG GGTGGCGAACCTTCCGAGTCTGGTAGCTCAGFP-\_E04.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAGTCAGGTGAATCTCCGGGTGGTTCCAGCGGTTCTGAGTCA1_051_ GSGGEPSESGSSGSEGSS GGTTCTGGTGGCGAACCATCTGAGTCTGGTAGCTCAGFP-\_F04.ab1 GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCCGGTCCAGGCGAGTCTTCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCALCW0401_053_ GESPGGSSGSESGESPGG GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCAGFP-\_H04.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCAGGTGAATCTCCTGGTGGTTCTAGCGGTTCTGAATCALCW0401_054_ GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATCTTCAGFP-\_A05.ab1 GSGGEPSESGSS GAAGGTTCTTCTGGTCCTGGTGAATCCTCAGGTTCCGGTGGCGAACCATCTGAATCTGGTAGCTCALCW0401_059_ GSGGEPSESGSSGSEGSS GGTTCTGGTGGCGAACCATCCGAATCTGGTAGCTCAGFP-\_D05.ab1 GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCTGGTCCTGGCGAATCTTCAGGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAATCAFile name Amino acid sequence Nucleotide sequenceLCW0401_060_ GSGGEPSESGSSGSSESG GGTTCCGGTGGTGAACCGTCCGAATCTGGTAGCTCAGFP-\_E05.ab1 SSEGGPGSGGEPSESGSS GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCAGGTTCCGGTGGTGAACCTTCTGAGTCTGGTAGCTCALCW0401_061_ GSSESGSSEGGPGSGGEP GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCAGFP-\_F05.ab1 SESGSSGSEGSSGPGESS GGTGGCGAACCATCTGAATCTGGTAGCTCAGGTAGCGAAGGTTCTTCCGGTCCGGGTGAATCTTCA1_063_ GSGGEPSESGSSGSEGSS GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCAGFP-\_H05.ab1 GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCAGGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCALCW0401_066_ GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCATCCGAGTCTGGTAGCTCAGFP-\_B06.ab1 SSEGGPGSGGEPSESGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCAGGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCALCW0401_067_ GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCAGFP-\_C06.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAATCAGGTGAATCTCCAGGTGGTTCTAGCGGTTCCGAATCALCW0401_069_ GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCATCTGAGTCTGGTAGCTCAGFP-\_D06.ab1 GESPGGSSGSES GGTTCCGGTGGCGAACCGTCCGAGTCTGGTAGCTCAGGTGAATCTCCGGGTGGTTCCAGCGGTTCCGAATCALCW0401_070_ GSEGSSGPGESSGSSESG GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATCCTCAGFP-\_E06.ab1 SSEGGPGSEGSSGPGESS GGTTCCTCCGAAAGCGGTTCTTCCGAAGGTGGTCCAGGTAGCGAAGGTTCTTCCGGTCCTGGTGAATCTTCALCW0401_078_ SSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGGTCCAGFP-\_F06.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAATCAGGTGAATCTCCTGGTGGCTCCAGCGGTTCCGAGTCA1_079_ GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTCTTCAGFP-\_G06.ab1 GPGESSGSGGEPSESGSS GGTAGCGAAGGTTCTTCCGGTCCTGGCGAGTCTTCAGGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA Example 2: uction ofXTEN_AE36 ts A codon y encoding XTEN sequences of 36 amino acid length was constructed. TheXTEN sequence was ated XTEN_AE36. Its segments have the amino acid sequence [X]3 where Xis a 12mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 23), GSEPATSGSETP (SEQ IDNO: 24), TPESGP (SEQ ID NO: 25), or GTSTEPSEGSAP (SEQ ID NO: 26). The insert wasobtained by annealing the following pairs of orylated synthetic oligonucleotide pairs:AElfor: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA (SEQ ID NO: 1628)AElreV: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT (SEQ ID NO: 1629)AE2for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC (SEQ ID NO: 1630)AE2reV: ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT (SEQ ID NO: 1631)AE3for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC (SEQ ID NO: 1632)AE3reV: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT (SEQ ID NO: 1633)AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC (SEQ ID NO: 1634)AE4reV: ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT (SEQ ID NO: 1635) We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylatedoligonucleotide pr_3KpnIstopperReV: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealedoligonucleotide pairs were ligated, which ed in a mixture of products with varying length thatrepresents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The productsponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gelelectrophoresis and ligated into the BsaI/Kpnl digested stuffer vector pCWO35 9. Most of the clones inthe resulting library designated LCWO402 showed green fluorescence after induction which shows thatthe sequence of XTEN_AE36 had been ligated in frame with the GFP gene and most sequences ofXTEN_AE36 show good expression.
We screened 96 isolates from library LCWO402 for high level of fluorescence by stamping themonto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 es wereidentified that contained segments with 36 amino acids as well as strong cence. These isolateswere sequenced and 37 clones were identified that contained correct XTEN_AE36 segments. The filenames of the nucleotide and amino acid constructs and the sequences for these segments are listed inTable l 4.
Table 14: DNA and Amino Acid ces for AE 36-mer motifs (SEQ ID NOS 279-352,res ectivel in order of a earanceFile name Amino acid seguence Nucleotide ceLCWO402_002_ GSPAGSPTSTEEGTSE GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGFP-\_A07.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGSAP GGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCALCWO402_003_ GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGFP-\_B07.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCAGSAP GGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCALCWO402_004_ GTSTEPSEGSAPGTSE GGTACCTCTACCGAACCGTCTGAAGGTAGCGCACCAGFP-\_C07.ab1 GPGTSESATP GGTACCTCTGAAAGCGCAACTCCTGAGTCCGGTCCAESGP GGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCALCWO402_005_ GTSTEPSEGSAPGTSE GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCAGFP-\_D07.ab1 SATPESGPGTSESATP GGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCAESGP GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCALCWO402_006_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAE07.ab1 SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCASTEE CCGGCAGGTTCTCCGACTTCCACTGAGGAALCWO402_008_ GTSESATPESGPGSEP TCTGAAAGCGCAACCCCTGAATCCGGTCCAF07.ab1 ATSGSETPGTSTEPSE GAACCGGCTACTTCTGGCTCTGAGACTCCAGSAP GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA2_009_ GSPAGSPTSTEEGSPA GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAGGAAG07.ab1 GSPTSTEEGSEPATSG GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAAGAASETP GGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCALCWO402_01 1_ GSPAGSPTSTEEGTSE GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGFP-\_A08.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGSAP GGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCALCWO402_012_ GSPAGSPTSTEEGSPA GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGFP-\_B08.ab1 GSPTSTEEGTSTEPSE GGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAAGSAP TCTACCGAACCTTCCGAAGGTAGCGCTCCALCWO402_013_ GTSESATPESGPGTST GGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGFP-\_C08.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGSAP GGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCALCWO402_014_ GTSTEPSEGSAPGSPA GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGFP-\_D08.ab1 GSPTSTEEGTSTEPSE GGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGSAP GGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCALCWO402_015_ GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAGFP-\_E08.ab1 GSPTSTEEGTSESATP GGTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAAESGP GGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCALCWO402_016_ GTSTEPSEGSAPGTSE GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGFP-\_F08.ab1 SATPESGPGTSESATP GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAWO 22617 2012/046326File name Amino acid sequence Nucleotide sequenceESGP GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCALCW0402_020_ GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCAGFP-\_G08.ab1 ATSGSETPGSPAGSPT GGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCASTEE GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAALCW0402_023_ GSPAGSPTSTEEGTSE GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAAGFP-\_A09.ab1 SATPESGPGSEPATSG GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCASETP GGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCALCW0402_024_ GTSESATPESGPGSPA TCTGAAAGCGCTACTCCTGAGTCCGGCCCAGFP-\_B09.ab1 GSPTSTEEGSPAGSPT GGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAASTEE GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAALCW0402_025_ GTSTEPSEGSAPGTSE GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGFP-\_C09.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGSAP GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCALCW0402_026_ GSPAGSPTSTEEGTST CCGGCAGGCTCTCCGACTTCCACCGAGGAAGFP-\_D09.ab1 EPSEGSAPGSEPATSG TCTACTGAACCTTCTGAGGGTAGCGCTCCASETP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCALCW0402_027_ GSPAGSPTSTEEGTST GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAE09.ab1 APGTSTEPSE GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCAGSAP GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCALCW0402_032_ GSEPATSGSETPGTSE GAACCTGCTACCTCCGGTTCTGAAACCCCAGFP-\_H09.ab1 SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCASTEE GGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAALCW0402_034_ GTSESATPESGPGTST GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGFP-\_A10.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGSAP GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCALCW0402_036_ GSPAGSPTSTEEGTST GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAAGFP-\_C10.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGSAP GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCALCW0402_039_ GTSTEPSEGSAPGTST TCTACCGAACCGTCCGAGGGCAGCGCTCCAGFP-\_E10.ab1 EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGSAP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCALCW0402_040_ GSEPATSGSETPGTSE GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAF10.ab1 SATPESGPGTSTEPSE GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGSAP GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCALCW0402_041_ GTSTEPSEGSAPGSPA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCAGFP-\_G10.ab1 GSPTSTEEGTSTEPSE GGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAAGSAP GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCALCW0402_050_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGFP-\_A11.ab1 SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCASETP GGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCALCW0402_051_ SGSETPGTSE GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCAGFP-\_B11.ab1 SATPESGPGSEPATSG TCTGAAAGCGCTACTCCTGAGTCTGGCCCASETP GGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCALCW0402_059_ GSEPATSGSETPGSEP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGFP-\_E11.ab1 ATSGSETPGTSTEPSE GGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCAGSAP GGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCALCW0402_060_ GTSESATPESGPGSEP GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCAGFP-\_F11.ab1 ATSGSETPGSEPATSG GGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCASETP GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCALCW0402_061_ GTSTEPSEGSAPGTST GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGFP-\_G11.ab1 EPSEGSAPGTSESATP TCTACCGAACCGTCCGAGGGCAGCGCACCAESGP TCTGAAAGCGCAACCCCTGAATCCGGTCCALCW0402_065_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGFP-\_A12.ab1 SATPESGPGTSESATP GGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCAESGP GGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCALCW0402_066_ GSEPATSGSETPGSEP GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCAGFP-\_B12.ab1 ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCAGSAP GGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCALCW0402_067_ GSEPATSGSETPGTST GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCAGFP-\_C12.ab1 EPSEGSAPGSEPATSG GGTACTTCTACCGAACCGTCCGAGGGTAGCGCTCCASETP GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCAFile name Amino acid sequence Nucleotide sequence2_069_ GTSTEPSEGSAPGTST GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGFP-N_D12.ab1 EPSEGSAPGSEPATSG GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCASETP GGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCALCW0402_073_ GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGFP-N_F12.ab1 TPGSPAGSPT GGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCASTEE GGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAALCW0402_074_ GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAGFP-N_G12.ab1 GSPTSTEEGTSESATP GGTAGCCCAGCTGGTTCTCCAACCTCTACTGAGGAAESGP GGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCALCW0402_075_ GTSESATPESGPGSEP GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGFP-N_H12.ab1 ATSGSETPGTSESATP GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAESGP TCTGAAAGCGCAACCCCGGAATCTGGTCCA Example 3: Construction ofXTEN_AF36 segments A codon y encoding sequences of 36 amino acid length was constructed. The sequenceswere designated XTEN_AF36. Its segments have the amino acid sequence [X]3 where X is a 12merpeptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 27), GTSTPESGSASP (SEQ ID NO: 28),GTSPSGESSTAP (SEQ ID NO: 29), or GSTSSTAESPGP (SEQ ID NO: 30). The insert was obtainedby annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:AF 1 for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC (SEQ ID NO: 1636)AP 1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA (SEQ ID NO: 1637)AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC (SEQ ID NO: 1638)AF2reV: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT (SEQ ID NO: 1639)AF3for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC (SEQ ID NO: 1640)AF3reV: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT (SEQ ID NO: 1641)AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC (SEQ ID NO: 1642)AF4reV: ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA (SEQ ID NO: 1643) We also annealed the phosphorylated ucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylatedoligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealedoligonucleotide pairs were ligated, which ed in a mixture of products with varying length thatrepresents the varying number of 12mer s ligated to one BbsI/KpnI segment The productscorresponding to the length of 36 amino acids were isolated from the mixture by preparative e gelelectrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCWO35 9. Most of the clones inthe resulting library designated LCWO403 showed green fluorescence after induction which shows thatthe sequence ofXTEN_AF36 had been ligated in frame with the GFP gene and most ces ofXTEN_AF36 show good expression.
We screened 96 isolates from library 3 for high level of fluorescence by stamping themonto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates werefied that contained segments with 36 amino acids as well as strong cence. These isolateswere sequenced and 44 clones were identified that contained correct XTEN_AF36 segments. The filenames of the nucleotide and amino acid constructs and the ces for these segments are listed inTable 15.
Table 15: DNA and Amino Acid Seguences for AF 36-mer motifs (SEQ ID NOS 353-440,res ectivel in order of a earanceFile name Amino acid sequence Nucleotide ceLCW0403_004_ GTSTPESGSASPGTSP GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCAGFP-\_A01.abl SGESSTAPGTSPSGES TCTCCTAGCGGTGAATCTTCTACTGCTCCAGSTAP GTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCALCW0403_005_ ESSTAPGSTS GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGFP-\_B01.abl STAESPGPGTSPSGES GGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAGSTAP GTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCALCW0403_006_ GSTSSTAESPGPGTSP GGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAGGFP-\_C01.abl SGESSTAPGTSTPESG GTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCAGGSASP TACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCALCW0403_007_ GSTSSTAESPGPGSTS ACCAGCTCTACTGCAGAATCTCCTGGCCCAGGFP-\_D01.abl STAESPGPGTSPSGES GTTCCACCAGCTCTACCGCAGAATCTCCGGGTCCAGSTAP GTACTTCCCCTAGCGGTGAATCTTCTACCGCACCALCW0403_008_ GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAGGFP-\_E01.abl SGESSTAPGTSTPESG GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGSASP TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCALCW0403_010_ GSTSSTAESPGPGTST GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAGGFP-\_F01.abl PESGSASPGSTSESPS GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGGTAP GTTCTACTAGCGAATCTCCTTCTGGCACTGCACCALCW0403_011_ GSTSSTAESPGPGTST GGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAGGFP-\_G01.abl PESGSASPGTSTPESG GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAGSASP CTACCCCTGAAAGCGGTTCTGCATCTCCALCW0403_012_ GSTSESPSGTAPGTSP GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGFP-\_H01.abl APGSTSESPS GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGGTAP TTCTACTAGCGAATCTCCTTCTGGCACTGCACCALCW0403_013_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCAGFP-\_A02.abl STAESPGPGTSPSGES GGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAGSTAP GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCALCW0403_014_ GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCAGAATCTCCTGGCCCAGGFP-\_B02.abl PESGSASPGSTSESPS GTACCTCTACCCCTGAAAGCGGCTCTGCATCTCCAGGTAP CCAGCGAATCCCCGTCTGGCACCGCACCALCW0403_015_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGFP-\_C02.abl STAESPGPGTSPSGES GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGGSTAP TACCTCCCCGAGCGGTGAATCTTCTACTGCACCALCW0403_017_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGFP-\_D02.abl ESPSGTAPGSTSSTAE GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGSPGP GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA3_018_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCAGAATCTCCTGGCCCAE02.abl STAESPGPGSTSSTAE GGTTCCACTAGCTCTACCGCTGAATCTCCTGGTCCAGSPGP GTTCTACTAGCTCTACCGCTGAATCTCCTGGTCCALCW0403_019_ PSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAGGFP-\_F02.abl STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCTGAATCTCCTGGCCCAGGSPGP TTCCACTAGCTCTACTGCAGAATCTCCTGGTCCALCW0403_023_ GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGFP-\_H02.abl ESPSGTAPGSTSESPS GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGGGTAP TTCTACCAGCGAATCTCCTTCTGGTACTGCACCALCW0403_024_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCTGAATCTCCTGGCCCAGGFP-\_A03.abl STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGGSPGP TTCCACCAGCTCTACCGCTGAATCTCCGGGTCCALCW0403_025_ GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACCGCAGAATCTCCTGGTCCAGB03.abl STAESPGPGTSPSGES GTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGGSTAP TACCTCCCCTAGCGGCGAATCTTCTACCGCTCCALCW0403_028_ GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAGGFP-\_D03.abl PSGATGSPGSSTPSGA GTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGGTGSP TAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAWO 22617 File name Amino acid sequence Nucleotide sequenceLCWO403_029_ GTSPSGESSTAPGTST GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGFP-\_E03.ab1 PESGSASPGSTSSTAE GTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAGSPGP GTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCALCWO403_030_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGFP-\_F03.ab1 STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGGSASP TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCALCWO403_031_ GTSPSGESSTAPGSTS TCTCCTAGCGGTGAATCTTCTACCGCTCCAGGFP-\_G03.ab1 STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCTGAATCTCCTGGCCCAGGSASP TACCCCGGAAAGCGGCTCCGCTTCTCCALCWO403_033 PSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCACCAGGFR\_H01mfi STAESPGPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGGSPGP TTCCACCAGCTCTACCGCAGAATCTCCTGGTCCALCWO403_035 AESPGPGSTS GGTTCCACCAGCTCTACCGCTGAATCTCCGGGCCCAGFR\_A04dfi ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCASPGP GGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCALCWO403_036 GSTSSTAESPGPGTSP GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAGGFPJ\_B04abT SGESSTAPGTSTPESG GTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAGSASP GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCALCWO403_039 GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGFPJ\_C04abT ESPSGTAPGTSPSGES GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGSTAP GTACTTCTCCTAGCGGCGAATCTTCTACCGCACCALCWO403_041 GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGFR\_D04dfi ESPSGTAPGTSTPESG GTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAGSASP GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCALCWO403_044 GTSTPESGSASPGSTS GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGFPJ\_E04abT STAESPGPGSTSSTAE CTAGCTCTACCGCAGAATCTCCGGGCCCAGSPGP GTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCALCWO403_046 GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAGFP:\_F04abT ESPSGTAPGTSPSGES GGTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAGSTAP GTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCALCWO403_047 GSTSSTAESPGPGSTS ACTAGCTCTACCGCTGAATCTCCTGGCCCAGo4mfi STAESPGPGSTSESPS GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAGGTAP GTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCALCWO403_049 GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCTGGCCCAGFR\_H04MH STAESPGPGTSTPESG GGTTCTACTAGCTCTACCGCAGAATCTCCTGGTCCAGSASP GTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCALCWO403_051 GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAG05dfi STAESPGPGSTSESPS GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGGGTAP TTCTACTAGCGAATCTCCTTCTGGTACCGCTCCALCWO403_053 GTSPSGESSTAPGSTS GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCAGFPJ\_BOSabT ESPSGTAPGSTSSTAE GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAGSPGP GTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCALCWO403_054 PSGTAPGTSP GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGFPJ\_C053bT SGESSTAPGSTSSTAE GTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGSPGP TTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA3_057 GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGFR\_D05dfi ESPSGTAPGTSPSGES GTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAGSTAP GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCALCWO403_058 GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAGGFPJ\_E053bT ESPSGTAPGTSTPESG GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAGSASP GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA3_060 GTSTPESGSASPGSTS GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAGFP:\_F05abT ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCASPGP GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCALCWO403_063 GSTSSTAESPGPGTSP ACTAGCTCTACTGCAGAATCTCCGGGCCCAGFR\_GOimfi SGESSTAPGTSPSGES GGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAGSTAP GTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCALCWO403_064_ ESSTAPGTSP GGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAGGFP-\_H05.ab1 SGESSTAPGTSPSGES GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGGSTAP TACCTCCCCTAGCGGTGAATCTTCTACCGCACCALCWO403_065_ GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAGFile name Amino acid sequence Nucleotide sequenceGFP-\_A06.ab1 PESGSASPGSTSESPS GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGGGTAP TTCTACTAGCGAATCTCCGTCTGGCACCGCACCALCW0403_066_ GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGB06.ab1 SGESSTAPGTSPSGES GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGSTAP TACTTCCCCTAGCGGCGAATCTTCTACCGCTCCALCW0403_067_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAGGFP-\_C06.ab1 PESGSASPGSTSSTAE GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGGSPGP TTCCACTAGCTCTACCGCTGAATCTCCGGGTCCALCW0403_068_ GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAGGFP-\_D06.ab1 STAESPGPGSTSESPS GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGGGTAP TTCTACCAGCGAATCTCCGTCTGGCACCGCACCALCW0403_069_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAGFP-\_E06.ab1 PESGSASPGTSTPESG GGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAGSASP GTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCALCW0403_070_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGFP-\_F06.ab1 PESGSASPGTSTPESG GTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGGSASP TACTCCGGAAAGCGGTTCTGCATCTCCA Example 4: Construction _AG36 segments A codon y encoding sequences of 36 amino acid length was constructed. The sequenceswere designated XTEN_AG36. Its segments have the amino acid sequence [X]3 where X is a 12merpeptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32),GSSPSASTGTGP (SEQ ID NO: 33), or GASPGTSSTGSP (SEQ ID NO: 34). The insert was obtainedby annealing the ing pairs of phosphorylated synthetic oligonucleotide pairs:AGlfor: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC (SEQ ID l\O: 1644)AGlreV: ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT (SEQ ID NO: 1645)AG2for: AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC (SEQ ID l\O: 1646)AG2reV: ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT (SEQ ID NO: 1647): TAGCCCKTCTGCWTCYACYGGTACYGGYCC (SEQ ID l\O: 1648)AG3reV: ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA (SEQ ID V0: 1649)AG4for: AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC (SEQ ID NO: 1650): ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC (SEQ ID V0: 1651) We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor:AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylatedoligonucleotide nIstopperRev: GTGAAGACGA (SEQ ID NO: 1627). The edoligonucleotide pairs were ligated, which resulted in a mixture of products with varying length thatrepresents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The productscorresponding to the length of 36 amino acids were isolated from the e by preparative agarose gelophoresis and ligated into the BsaI/KpnI digested stuffer vector pCWO35 9. Most of the clones inthe resulting library designated LCWO404 showed green fluorescence after induction which shows thatthe sequence ofXTEN_AG36 had been ligated in frame with the GFP gene and most sequences ofXTEN_AG36 show good expression.
We screened 96 isolates from library LCWO404 for high level of fluorescence by stamping themonto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates werefied that contained segments with 36 amino acids as well as strong fluorescence. These isolateswere sequenced and 44 clones were identified that contained correct XTEN_AG36 segments. The filenames of the nucleotide and amino acid constructs and the sequences for these segments are listed inTable 16.
Table 16: DNA and Amino Acid ces for AG 36-mer motifs (SEQ ID NOS 8,res ectivel in order of a earanceFile name Amino acid sequence Nucleotide sequence4_001_ SSTGSPGTPGS TCCCCGGGCACTAGCTCTACCGGTTCTCCAA07.ab1 GTASSSPGSSTPSGATG GGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAGSP GTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCALCW0404_003_ GATGSPGSSPS GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGFP-\_B07.ab1 ASTGTGPGSSTPSGATG GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGGSP TAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA4_006_ GASPGTSSTGSPGSSPS GGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGFP-\_C07.ab1 ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGSP TAGCTCTACCCCGTCTGGTGCTACTGGTTCCCCALCW0404_007_ GTPGSGTASSSPGSSTPS GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGFP-\_D07.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCTTCTGGTGCAACTGGTTCCCCAGGP TGCATCCCCTGGTACTAGCTCTACCGGTTCTCCALCW0404_009_ GTPGSGTASSSPGASPG GGTACCCCTGGCAGCGGTACTGCTTCTTCTTCTCCAGGFP-\_E07.ab1 TSSTGSPGSRPSASTGT GTGCTTCCCCTGGTACCAGCTCTACCGGTTCTCCAGGGP TTCTAGACCTTCTGCATCCACCGGTACTGGTCCALCW0404_01 1_ GASPGTSSTGSPGSSTPS GGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGFP-\_F07.ab1 GATGSPGASPGTSSTGS GTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGP TGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCALCW0404_012_ GTPGSGTASSSPGSSTPS GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGFP-\_G07.ab1 GATGSPGSSTPSGATGS GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGP GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCALCW0404_014_ GASPGTSSTGSPGASPG GGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAGGFP-\_H07.ab1 TSSTGSPGASPGTSSTGS GTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGGP TGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCALCW0404_015_ GSSTPSGATGSPGSSPS GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCAGFP-\_A08.ab1 PGASPGTSSTG GGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAGSP GTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCALCW0404_016_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCTTCTGGTGCTACCGGTTCCCCAGGFP-\_B08.ab1 GATGSPGTPGSGTASSS CTACTCCTTCTGGTGCTACTGGTTCCCCAGGP TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCALCW0404_017_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGFP-\_C08.ab1 GATGSPGASPGTSSTGS GTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGGP TGCATCCCCTGGCACCAGCTCTACCGGTTCTCCALCW0404_0 1 8_ GTPGSGTASSSPGSSPS CCTGGTAGCGGTACCGCATCTTCCTCTCCAGGFP-\_D08.ab1 ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCATCTACCGGTACCGGTCCAGGSP TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCALCW0404_023_ GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGFP-\_F08.ab1 ASTGTGPGTPGSGTASS GTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGSP TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCALCW0404_025_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGFP-\_G08.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGP TGCTTCTCCGGGTACCAGCTCTACTGGTTCTCCALCW0404_029_ GTPGSGTASSSPGSSTPS GGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAGGFP-\_A09.ab1 GATGSPGSSPSASTGTG GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGP TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCALCW0404_030_ GSSTPSGATGSPGTPGS GGTAGCTCTACTCCTTCTGGTGCAACCGGCTCCCCAGGFP-\_B09.ab1 GTASSSPGTPGSGTASS CGGGCAGCGGTACCGCATCTTCCTCTCCAGSP GTACTCCGGGTAGCGGTACTGCTTCTTCTTCTCCALCW0404_03 1_ TASSSPGSSTPS GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAGGFP-\_C09.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGG 2012/046326File name Amino acid sequence Nucleotide ceP TGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCALCW0404_034_ GSSTPSGATGSPGSSTPS GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGGFP-\_D09.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGP CCCCGGGTACTAGCTCTACCGGTTCTCCALCW0404_035_ GASPGTSSTGSPGTPGS GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGFP-\_E09.ab1 GTASSSPGSSTPSGATG GTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAGSP GTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCALCW0404_036_ GSSPSASTGTGPGSSTPS GGTTCTAGCCCGTCTGCTTCCACCGGTACTGGCCCAGGFP-\_F09.ab1 GTPGSGTASSS GTAGCTCTACCCCGTCTGGTGCAACTGGTTCCCCAGGP TACCCCTGGTAGCGGTACCGCTTCTTCTTCTCCA4_037_ GASPGTSSTGSPGSSPS GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGFP-\_G09.ab1 ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGSP TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCALCW0404_040_ GASPGTSSTGSPGSSTPS TCCCCGGGCACCAGCTCTACCGGTTCTCCAGFP-\_H09.ab1 GATGSPGSSTPSGATGS GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAGP GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCALCW0404_041_ GTPGSGTASSSPGSSTPS GGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAGGFP-\_A10.ab1 GATGSPGTPGSGTASSS GTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGGP TACCCCGGGTAGCGGTACCGCATCTTCTTCTCCALCW0404_043_ GSSPSASTGTGPGSSTPS GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGGFP-\_C10.ab1 GATGSPGSSTPSGATGS GTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGP TAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCA4_045_ GASPGTSSTGSPGSSPS GGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAGGFP-\_D10.ab1 ASTGTGPGSSPSASTGT GTTCTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGGGP TTCTAGCCCTTCTGCATCCACTGGTACTGGTCCALCW0404_047_ GTPGSGTASSSPGASPG GGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAGGFP-\_F10.ab1 TSSTGSPGASPGTSSTGS GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGP TGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCALCW0404_048_ GSSTPSGATGSPGASPG GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGFP-\_G10.ab1 PGSSTPSGATGS GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGGP TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCALCW0404_049_ GSSTPSGATGSPGTPGS GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGFP-\_H10.ab1 GTASSSPGSSTPSGATG CGGGCAGCGGTACTGCTTCTTCCTCTCCAGGSP TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCALCW0404_050_ GASPGTSSTGSPGSSPS GGTGCATCTCCTGGTACCAGCTCTACTGGTTCTCCAGGFP-\_A11.ab1 ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGSP TAGCTCTACTCCTTCTGGTGCTACCGGTTCTCCALCW0404_051_ GSSTPSGATGSPGSSTPS GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGGFP-\_B11.ab1 GATGSPGSSTPSGATGS GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGGP TAGCTCTACCCCGTCTGGTGCAACTGGCTCTCCALCW0404_052_ GASPGTSSTGSPGTPGS GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAGFP-\_C11.ab1 GTASSSPGASPGTSSTG GGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAGSP GTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA4_053_ GSSTPSGATGSPGSSPS GGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAGGFP-\_D11.ab1 ASTGTGPGASPGTSSTG GTTCTAGCCCGTCTGCATCCACTGGTACCGGTCCAGGSP TGCTTCCCCTGGCACCAGCTCTACCGGTTCTCCALCW0404_057_ SSTGSPGSSTPS GGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGFP-\_E11.ab1 GATGSPGSSPSASTGTG CTACTCCGTCTGGTGCAACCGGCTCTCCAGGP TTCTAGCCCTTCTGCATCTACCGGTACTGGTCCALCW0404_060_ GTPGSGTASSSPGSSTPS GGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAGGFP-\_F11.ab1 GATGSPGASPGTSSTGS CTACTCCGTCTGGTGCAACTGGTTCCCCAGGP TGCTTCTCCGGGTACCAGCTCTACCGGTTCTCCALCW0404_062_ GSSTPSGATGSPGTPGS GGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAGFP-\_G11.ab1 GTASSSPGSSTPSGATG GGTACTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAGSP GTAGCTCTACTCCGTCTGGTGCTACCGGCTCCCCALCW0404_066_ GSSPSASTGTGPGSSPS GGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGGFP-\_H11.ab1 ASTGTGPGASPGTSSTG GTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGSP TGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCALCW0404_067_ TASSSPGSSTPS GGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAGGFP-\_A12.ab1 GATGSPGSNPSASTGTG GTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGGP TTCTAACCCTTCTGCATCCACCGGTACCGGCCCA 2012/046326File name Amino acid sequence Nucleotide sequenceLCWO404_068_ GSSPSASTGTGPGSSTPS GGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGFP-\_B12.ab1 GATGSPGASPGTSSTGS GTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGP TGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCALCWO404_069_ GSSTPSGATGSPGASPG GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGFP-\_C12.ab1 TSSTGSPGTPGSGTASSS CCCCGGGTACCAGCTCTACCGGTTCTCCAGP GTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCALCWO404_070_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAGGFP-\_D12.ab1 GSSTPSGATGS CTACCCCTTCTGGTGCAACCGGCTCCCCAGGP TAGCTCTACCCCTTCTGGTGCAACTGGCTCTCCALCWO404_073_ GASPGTSSTGSPGTPGS GGTGCTTCTCCTGGCACTAGCTCTACCGGTTCTCCAGGFP-\_E12.ab1 GTASSSPGSSTPSGATG GTACCCCTGGTAGCGGTACCGCATCTTCCTCTCCAGGSP TAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCALCWO404_075_ GSSTPSGATGSPGSSPS GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAGF12.ab1 ASTGTGPGSSPSASTGT GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGGGP TTCTAGCCCGTCTGCATCTACTGGTACTGGTCCALCWO404_080_ GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAGGFP-\_G12.ab1 ASTGTGPGSSPSASTGT GTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGGGP TTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCALCWO404_081_ GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCAGGFP-\_H12.ab1 ASTGTGPGTPGSGTASS GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGGSP TACCCCTGGCAGCGGTACCGCATCTTCCTCTCCA Example 5: Construction ofXTEN_AE864 XTEN_AE864 was constructed from serial dimerization ofXTEN_AE36 to AE72, 144, 288,576 and 864. A collection ofXTEN_AE72 segments was constructed from 37 different segments ofXTEN_AE36. Cultures of E. coli harboring all 37 different 36-amino acid segments were mixed andplasmid was isolated. This plasmid pool was digested with Bsal/Ncol to generate the small fragment asthe insert. The same plasmid pool was digested with Bbsl/Ncol to generate the large fragment as thevector. The insert and vector fragments were d ing in a doubling of the length and the ligationmixture was transformed into ld(DE3) cells to obtain colonies of XTEN_AE72.
This library of XTEN_AE72 segments was designated LCWO406. All clones from LCWO406were combined and dimerized again using the same process as bed above yielding libraryLCWO410 ofXTEN_AE144. All clones from LCWO410 were combined and dimerized again using thesame s as described above yielding library LCWO414 of E288. Two esLCWO414.001 and LCWO414.002 were randomly picked from the library and ced to verify theidentities. All clones from LCWO414 were combined and dimerized again using the same process asdescribed above yielding library LCWO418 ofXTEN_AES76. We screened 96 es from library8 for high level of GFP fluorescence. 8 isolates with right sizes of inserts by PCR and strongfluorescence were sequenced and 2 es (LCWO418.018 and LCWO418.052) were chosen for futureuse based on sequencing and expression data.
The specific clone pCWO432 ofXTEN_AE864 was constructed by combining LCWO418.018of XTEN_AE576 and LCWO414.002 ofXTEN_AE288 using the same dimerization process as describedabove.
Example 6: Construction ofXTEN_AM144 A collection ofXTEN_AM144 segments was constructed starting from 37 ent segments ofXTEN_AE36, 44 segments ofXTEN_AF36, and 44 ts ofXTEN_AG36.
Cultures of E. coli that harboring all 125 different 36-amino acid segments were mixed andplasmid was isolated. This plasmid pool was digested with BsaI/NcoI to generate the small fragment asthe . The same d pool was ed with BbsI/NcoI to generate the large fragment as thevector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligationmixture was transformed into BL21Gold(DE3) cells to obtain colonies of XTEN_AM72.
This library of XTEN_AM72 segments was ated LCWO461. All clones from LCWO461were combined and dimerized again using the same process as described above yielding libraryLCWO462. 1512 Isolates from library LCWO462 were screened for protein expression. Individualcolonies were transferred into 96 well plates and cultured overnight as starter cultures. These startercultures were diluted into fresh autoinduction medium and cultured for 20-3 Oh. Expression was measuredusing a fluorescence plate reader with excitation at 395 nm and emission at 510 nm. 192 isolates showedhigh level expression and were submitted for DNA sequencing. Most clones in library LCWO462 showedgood sion and similar physicochemical properties suggesting that most ations ofXTEN_AM36 segments yield useful XTEN sequences. Thirty isolates from LCWO462 were chosen as apreferred collection of XTEN_AM144 segments for the construction of unctional ns thatcontain multiple XTEN segments. The file names of the nucleotide and amino acid constructs and thesequences for these segments are listed in Table 17.
Table 17: DNA and amino acid seguences for AM144 segments [SEQ ID NOS 529-594,res ectivel in order of a earanceClone Sequence Trimmed n Sequence_r1 GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGC GTPGSGTASSSPGSTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCC STPSGATGSPGSSTPGTGCAACCGGCTCCCCAGGTAGCCCGGCTGGCTCTC SGATGSPGSPAGSPCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG TSTEEGTSESATPESAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCG GPGTSTEPSEGSAPCTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGG GSSPSASTGTGPGSTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT GTGPGASPCCGGGTACTAGCTCTACTGGTTCTCCAGGTACCTCTACCGAAC SPGTSTEPSCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTG EGSAPGTSTEPSEGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTG SAPGSEPATSGSETPAAACTCCA_r5 GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAGGTTCTA GSTSESPSGTAPGSTCTAGCGAATCCCCTTCTGGTACCGCACCAGGTACTTCTCCGAG SESPSGTAPGTSPSGCGGCGAATCTTCTACTGCTCCAGGTACCTCTACTGAACCTTCC ESSTAPGTSTEPSEGGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGC SAPGTSTEPSEGSAPAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGT GTSESATPESGPGACCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTA SPGTSSTGSPGSSTPGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCC SGATGSPGASPGTSGGGTACCAGCTCTACCGGTTCTCCAGGTTCTACTAGCGAATCT STGSPGSTSESPSGTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTG APGSTSESPSGTAPGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTT GTSTPESGSASPCTCCALCW462_r9 GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACT GTSTEPSEGSAPGTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA SESATPESGPGTSESAGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTT ATPESGPGTSTEPSEClone Sequence Trimmed Protein SequenceCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG GSAPGTSESATPESAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG GPGTSTEPSEGSAPCACCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAG GTSTEPSEGSAPGSGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCC SETPGSPACGGCTGGCTCTCCGACCTCCACCGAGGAAGGTGCTTCTCCTG GSPTSTEEGASPGTGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCTTC SSTGSPGSSPSASTGTACCGGTACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGGT TGPGSSPSASTGTGACTGGTCCA PLCW462_r10 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC GSEPATSGSETPGTTCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA SESATPESGPGTSESAGCGCTACTCCGGAATCCGGTCCAGGTTCTACCAGCGAATCT ATPESGPGSTSESPSCCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTG GTAPGSTSESPSGTGTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGC APGTSPSGESSTAPACCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGT GASPGTSSTGSPGSTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCTA SPSASTGTGPGSSTPCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCGTC SGATGSPGSSTPSGAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCT ATGSPGSSTPSGATACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTT GSPGASPGTSSTGSCTCCA P_r15 GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTA GASPGTSSTGSPGSGCCCTTCTGCATCCACCGGTACCGGTCCAGGTAGCTCTACCCC SPSASTGTGPGSSTPTTCTGGTGCAACCGGCTCTCCAGGTACTTCTGAAAGCGCTACC SGATGSPGTSESATCCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT SEPATSGSEGAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT TPGSEPATSGSETPCCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGT GTSESATPESGPGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCT GSAPGTSTEACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCTACCGAA PSEGSAPGTSTEPSECCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCT STEPSEGSGAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCT APGSEPATSGSETPGAAACTCCALCW462_r16 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC GTSTEPSEGSAPGSPCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG AGSPTSTEEGTSTEPAACCTTCTGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAA SEGSAPGTSESATPCTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCT ESGPGSEPATSGSECTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTG TPGTSESATPESGPGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG GSPAGSPTSTEEGTGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC SESATPESGPGTSTETACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGC PGSEPATSTACTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCC GSETPGTSTEPSEGSGAGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCT APGSEPATSGSETPGAAACTCCALCW462_r20 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGTTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC STEPSEGSAPGTSTEGAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCG PSEGSAPGTSTEPSETCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAG GSAPGTSTEPSEGSGGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGC APGTSTEPSEGSAPGCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA GTSTEPSEGSAPGTGGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACT SESATPESGPGTSESTCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTACTG ATPESGPGTSTEPSEAACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTT GSAPGSEPATSGSECTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCT GSPTSTEECCACCGAGGAALCW462_r23 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT SEGSAPGTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTEAACCTTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCC PSEGSAPGSTSESPSCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGG GTAPGSTSESPSGTCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCT APGTSTPESGSASPCCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT GSEPATSGSETPGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA ESGPGTSTECTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTACTGAAC PSEGSAPGTSTEPSECGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCC GSAPGTSESATPESClone Sequence Trimmed n SequenceCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT GPGTSESATPESGPCCGGCCCALCW462_r24 GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGTTCTA GSSTPSGATGSPGSGCCCGTCTGCTTCTACCGGTACCGGTCCAGGTAGCTCTACCCC SPSASTGTGPGSSTPTTCTGGTGCTACTGGTTCTCCAGGTAGCCCTGCTGGCTCTCCG SGATGSPGSPAGSPACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA SPAGSPTSTCTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTC EEGTSTEPSEGSAPCAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTC GASPGTSSTGSPGSTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGC SPSASTGTGPGTPGAGCGGTACTGCTTCTTCCTCTCCAGGTTCTACTAGCTCTACTG SGTASSSPGSTSSTACTGAATCTCCTGGCCCAGGTACTTCTCCTAGCGGTGAATCTTC ESPGPGTSPSGESSTTACCGCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCT APGTSTPESGSASPLCW462_r27 GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT SESATPESGPGTSTEGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCG PSEGSAPGTSTEPSETCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCG GSAPGTSESATPESGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCC SATPESGPGGCCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG GTPGSGTASSSPGACTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTC SPGTSSTGSPGASPTCCGGGCACTAGCTCTACTGGTTCTCCAGGTAGCCCTGCTGGC GTSSTGSPGSPAGSTCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCG PTSTEEGSPAGSPTSACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGT TEEGTSTEPSEGSAPAGCGCTCCALCW462_r28 GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACT GSPAGSPTSTEEGTTCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACT STEPSEGSAPGTSTEGAACCTTCTGAGGGCAGCGCTCCAGGTACCTCTACCGAACCG PSEGSAPGTSTEPSETCTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCT GSAPGTSESATPESGAGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCT GPGTSESATPESGPGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG GTPGSGTASSSPGSGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTC STPSGATGSPGASPTCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCTCTACTGAA GTSSTGSPGTSTEPSCCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC EGSAPGTSESATPECCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGT TEPSEGSAPAGCGCACCALCW462_r3 8 GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACT GSEPATSGSETPGTTCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCG ESGPGSEPAGCTACTTCCGGCTCTGAAACCCCAGGTAGCTCTACCCCGTCTG TSGSETPGSSTPSGAGTGCAACCGGCTCCCCAGGTACTCCTGGTAGCGGTACCGCTT TGSPGTPGSGTASSCTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTC SPGSSTPSGATGSPCCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGT GASPGTSSTGSPGSAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC STPSGATGSPGASPCGGGTACCAGCTCTACCGGTTCTCCAGGTAGCGAACCTGCTA SPGSEPATSCTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCCGA GSETPGTSTEPSEGSGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA APGSEPATSGSETPAACTCCALCW462_r39 GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGTTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAA STEPSEGSAPGTSESAGCGCAACCCCTGAATCCGGTCCAGGTAGCCCTGCTGGCTCT ATPESGPGSPAGSPCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACT TSTEEGSPAGSPTSTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGC EEGTSTEPSEGSAPGCTCCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GSPAGSPTSTEEGTGGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC STEPSEGSAPGTSTETCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTGCTTCCCCG PSEGSAPGASPGTSAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTT STGSPGSSPSASTGTCTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGG SASTGTGPTACTGGTCCALCW462_r41 GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTT GSSTPSGATGSPGACTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCC SPGTSSTGSPGSSTPGTCTGGTGCTACTGGCTCTCCAGGTAGCCCTGCTGGCTCTCCA PGSPAGSPACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA TSTEEGTSESATPESClone ce Trimmed Protein SequenceTCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC GPGSEPATSGSETPCCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTA GASPGTSSTGSPGSGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCC STPSGATGSPGSSPSTTCTGCATCTACCGGTACTGGTCCAGGTTCTACCAGCGAATCC ASTGTGPGSTSESPSCCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTG GTAPGSTSESPSGTGCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT APGTSTPESGSASPCTCCALCW462_r42 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTA GSTSESPSGTAPGSTCTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAG SESPSGTAPGTSPSGATCTTCTACCGCACCAGGTACCTCTGAAAGCGCTAC ESSTAPGTSESATPETCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGG SGPGTSTEPSEGSAPTAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGC GTSTEPSEGSAPGTACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG STEPSEGSAPGTSESTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT ATPESGPGTSTEPSEACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCTCTACCCCG GSAPGSSTPSGATGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT SPGASPGTSSTGSPCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG GSSTPSGATGSPCTCTCCA_r43 ACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCT GSTSSTAESPGPGTSCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAG PSGESSTAPGTSPSGCGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACCGCT GSTSSTAESGAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTC PGPGSTSSTAESPGPCTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCC GTSTPESGSASPGTSAGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTTCT PSGESSTAPGSTSSTACCAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACCC GTSTPESGSCGGAAAGCGGCTCCGCTTCTCCAGGTTCTACCAGCTCTACCG ASPGSTSSTAESPGPCTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGG GSTSESPSGTAPGTSCACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCA PSGESSTAPLCW462_r45 GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTTCTA GTSTPESGSASPGSTCCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCT SESPSGTAPGSTSSTCTACTGCTGAATCTCCGGGCCCAGGTACCTCTACTGAACCTTC AESPGPGTSTEPSECGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGG GSAPGTSTEPSEGSCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG APGTSESATPESGPTCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGG GTSESATPESGPGTTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCT STEPSEGSAPGTSTEACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGC PGTSESATPGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCC ESGPGTSTEPSEGSGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGT APGTSTEPSEGSAPAGCGCTCCC_r47 TCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC GTSTEPSEGSAPGTTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCG STEPSEGSAPGSEPAGCAACCTCCGGTTCTGAAACTCCAGGTACTTCTACTGAACCGT TSGSETPGTSTEPSECTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG GSAPGTSESATPESAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCG GPGTSESATPESGPGCCCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG GASPGTSSTGSPGSGTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC GTGPGSSTPTACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCG PGSSTPSGTCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTG ATGSPGSSTPSGATCTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCG GSPGASPGTSSTGSGTTCTCCA PLCW462_r54 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGC GSEPATSGSETPGSGAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACT EPATSGSETPGTSTEGAACCTTCTGAGGGCAGCGCACCAGGTAGCGAACCTGCAACC PSEGSAPGSEPATSTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GSETPGTSESATPESGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GPGTSTEPSEGSAPGCACCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG GSSTPSGATGSPGSGTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTC STPSGATGSPGASPTCCGGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACCCCG GTSSTGSPGSSTPSGTCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT ATGSPGASPGTSSTCTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG GSPGSSTPSGATGSClone Sequence Trimmed Protein SequenceCTCTCCA PLCW462_r55 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG GSAPGTSTECCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCTA PSEGSAPGTSESATPCTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCCGAAG ESGPGTSTEPSEGSGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCG APGTSTEPSEGSAPCTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGG PSGTAPGTSTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCC PSGESSTAPGTSPSGCCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCCCGGCTGGC ESSTAPGSPAGSPTSTCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC TEEGTSESATPESGPCTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA GTSTEPSEGSAPGCGCTCCALCW462_r57 GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGC GTSTEPSEGSAPGSGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCT EPATSGSETPGSPACCGACCTCCACCGAGGAAGGTAGCCCGGCAGGCTCT GSPTSTEEGSPAGSPCCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG TSESATPESGAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGC GPGTSTEPSEGSAPGCACCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA GTSTEPSEGSAPGTGGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACT STEPSEGSAPGTSESTCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCTCTACT ATPESGPGSSTPSGCCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTT ATGSPGSSPSASTGCCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTA TGPGASPGTSSTGSCTGGTTCTCCA PLCW462_r61 GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAGGTAGC GSEPATSGSETPGSPCCTGCTGGCTCTCCGACCTCTACCGAAGAAGGTACCTCTGAA AGSPTSTEEGTSESAGCGCTACCCCTGAGTCTGGCCCAGGTACCTCTACTGAACCTT PGTSTEPSECCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG GSAPGTSTEPSEGSGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG APGTSESATPESGPGTCCAGGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAG GTSTPESGSASPGSTGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTA TAPGSTSSTCTAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCTGAAA AESPGPGTSESATPGCGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGT STEPSEGSCCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG APGTSTEPSEGSAPGTAGCGCTCCALCW462_r64 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGTTCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTEAACCTTCCGAAGGTAGCGCACCAGGTACCTCTACCGAACCGT PSEGSAPGTSTEPSECTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTG GSAPGTSESATPESAGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCTG GPGTSESATPESGPGCCCAGGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG TASSSPGSGTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTC STPSGATGSPGASPTCCGGGTACCAGCTCTACCGGTTCTCCAGGTTCCACCAGCTCT GTSSTGSPGSTSSTAACTGCTGAATCTCCTGGTCCAGGTACCTCTCCTAGCGGTGAAT ESPGPGTSPSGESSTCTTCTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGC APGTSTPESGSASPTTCTCCALCW462_r67 GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACT GSPAGSPTSTEEGTTCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACC SESATPESGPGTSTEGAACCGTCTGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA PSEGSAPGTSESATPACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGC ESGPGSEPATSGSETCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC TPGTSTEPSEGSAPGCACCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GSPAGSPTSTEEGTGGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC STEPSEGSAPGTSTETCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACTTCTACC PSEGSAPGTSTEPSETCCGAGGGCAGCGCTCCAGGTACTTCTACTGAACCT GSAPGTSTEPSEGSTCTGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAA APGTSTEPSEGSAPGGTAGCGCACCALCW462_r69 GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTA GTSPSGESSTAPGSTCTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAG SSTAESPGPGTSPSGCGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACT GTSESATPECCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGT SGPGTSTEPSEGSAPAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA GTSTEPSEGSAPGSSClone Sequence Trimmed n SequenceCCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTA PSASTGTGPGSSTPSGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCC GATGSPGASPGTSSGGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAA TGSPGTSTPESGSASAGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTT PGTSPSGESSTAPGTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGC SPSGESSTAPTCCALCW462_r70 GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC GTSESATPESGPGTTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTEAACCGTCCGAAGGTAGCGCACCAGGTAGCCCTGCTGGCTCTC PSEGSAPGSPAGSPCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTT TSTEEGSPAGSPTSTAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCG EEGTSTEPSEGSAPCTCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGG STGTGPGSTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCT STPSGATGSPGSSTPACTCCTTCTGGTGCAACTGGCTCTCCAGGTAGCGAACCGGCA SGATGSPGSEPATSACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACT GSETPGTSESATPESCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCT GPGSEPATSGSETPGAAACCCCALCW462_r72 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGTTCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC STEPSEGSAPGTSTETCTGAAGGTAGCGCACCAGGTAGCTCTACCCCGTCT PSEGSAPGSSTPSGGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTA ATGSPGASPGTSSTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTC GSPGSSTPSGATGSTCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGG PGTSESATPESGPGSTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCT EPATSGSETPGTSTEACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACTAGCGAA PSEGSAPGSTSESPSTCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGT GTAPGSTSESPSGTCTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCG APGTSTPESGSASPCTTCTCCALCW462_r73 GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCA GTSTPESGSASPGSTCTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTC SSTAESPGPGSTSSTTACTGCTGAATCTCCTGGCCCAGGTTCTAGCCCTTCTGCATCT AESPGPGSSPSASTACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCG GTGPGSSTPSGATGCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCC GTSSTGSPAGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTAC GSEPATSGSETPGTCTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGC SESATPESGPGSPAAGGTTCTCCGACTTCCACTGAGGAAGGTTCTACTAGCGAATC GSPTSTEEGSTSESPTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCT SGTAPGSTSESPSGTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCT APGTSTPESGSASPTCTCCCLCW462_r78 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACT GSPAGSPTSTEEGTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SESATPESGPGTSTEAACCGTCCGAAGGTAGCGCTCCAGGTTCTACCAGCGAATCTC PSEGSAPGSTSESPSCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGG GTAPGSTSESPSGTACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCA APGTSPSGESSTAPACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT GTSTEPSEGSAPGSPAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCT AGSPTSTEEGTSTEPACCGAACCTTCTGAGGGTAGCGCACCAGGTAGCGAACCTGCA SEGSAPGSEPATSGGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT SETPGTSESATPESGCCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC PGTSTEPSEGSAPLCW462_r79 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC GTSTEPSEGSAPGSPCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG AGSPTSTEEGTSTEPAACCTTCTGAGGGTAGCGCACCAGGTACCTCCCCTAGCGGCG SEGSAPGTSPSGESSAATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTC TAPGTSPSGESSTAPTACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCA GTSPSGESSTAPGSTCCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTT SESPSGTAPGSTSESCTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTAC PSGTAPGTSTPESGSCCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCGAACCTGCAAC ASPGSEPATSGSETPCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GTSESATPESGPGTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC STEPSEGSAPGCACCAClone Sequence Trimmed Protein SequenceLCW462_r87 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC GSEPATSGSETPGTAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA SESATPESGPGTSESAGCGCTACTCCGGAATCCGGTCCAGGTACTTCTCCGAGCGGT ATPESGPGTSPSGESGAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAAT STAPGSTSSTAESPGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGC PGTSPSGESSTAPGSTCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGT TSESPSGTAPGTSPSACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCA GESSTAPGSTSSTAGCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACTCCGTC ESPGPGSSTPSGATTGGTGCAACCGGTTCCCCAGGTAGCTCTACCCCTTCTGGTGCA GSPGSSTPSGATGSACCGGCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAAACTGG PGSSTPSGANWLSCTCTCCLCW462_r88 GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGC GSPAGSPTSTEEGSPCCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCG AGSPTSTEEGTSTEPAACCTTCCGAAGGTAGCGCTCCAGGTACCTCTACTGAACCTT SEGSAPGTSTEPSECCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG STEPSEGSGCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG APGTSESATPESGPGTCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGG GASPGTSSTGSPGSTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCC STPSGATGSPGASPCCGGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGT SPGSSTPSGCTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG ATGSPGTPGSGTASCTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGG SSPGSSTPSGATGSPCTCTCCALCW462_r89 GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTC GSSTPSGATGSPGTCGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC PGSGTASSSPGSSTPTTCTGGTGCTACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCT SGATGSPGSPAGSPACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAG TSTEEGTSESATPESTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT GPGTSTEPSEGSAPCCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGT GTSESATPESGPGSAGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT EPATSGSETPGTSESGAAAGCGCAACCCCGGAATCTGGTCCAGGTACTTCTACTGAA ATPESGPGTSTEPSECCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC GSAPGTSESATPESCCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAG GPGTSESATPESGPTCCGGCCCA Example 7: Construction ofXTEN_AM288] The entire library LCWO462 was dimerized as described in Example 6 resulting in a library ofXTEN_AM288 clones ated LCWO463. 1512 isolates from library LCWO463 were screened usingthe protocol described in Example 6. 176 highly expressing clones were sequenced and 40 preferredM288 segments were chosen for the construction of multifunctional proteins that containmultiple XTEN ts with 288 amino acid residues.
Example 8: Construction ofXTEN_AM432 We ted a library ofXTEN_AM432 segments by ining segments from library2 _AM144 segments and segments from library LCWO463 ofXTEN_AM288ts. This new library ofXTEN_AM432 segment was designated LCWO464. Plasmids wereisolated from cultures of E. coli harboring LCWO462 and LCWO463, tively. 1512 isolates fromlibrary LCWO464 were screened using the protocol described in Example 6. 176 highly expressingclones were sequenced and 39 preferred XTEN_AM432 segment were chosen for the construction oflonger XTENs and for the construction of multifunctional proteins that contain multiple XTEN segmentswith 432 amino acid residues.
In parallel we constructed library LMSOlOO ofXTEN_AM432 segments using preferredsegments ofXTEN_AM144 and M288. Screening this library yielded 4 isolates that wereselected for further construction e 9: uction ofXTEN_AM875 The stuffer vector pCWO359 was digested with BsaI and KpnI to remove the stuffer tand the resulting vector fragment was isolated by agarose gel purification.
We annealed the phosphorylated ucleotide BsaI-AscI-KpnIforP:AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQID NO: 1652) and the non-phosphorylated oligonucleotide BsaI-AscI-KpnIrev:CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC (SEQ ID NO: 1653)for introducing the sequencing island A (SI-A) which encodes amino acids GASASGAPSTG (SEQ IDNO: 1654) and has the restriction enzyme AscI recognition nucleotide sequence GGCGCGCC inside.
The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCWO359ed above to yield pCWO466 containing SI-A. We then generated a y ofXTEN_AM443ts by recombining 43 preferred XTEN_AM432 segments from e 8 and SI-A segmentsfrom pCWO466 at C-terminus using the same dimerization process described in Example 5. This newlibrary of XTEN_AM443 segments was designated LCWO479.
We generated a library ofXTEN_AM875 segments by recombining segments from libraryLCWO479 ofXTEN_AM443 segments and 43 preferred M432 segments from Example 8using the same dimerization s described in example 5. This new library of XTEN_AM875 segmentwas designated LCWO481.
] Example 10: Construction ofXTEN_AM1318 We annealed the phosphorylated oligonucleotide BsaI-FseI-KpnIforP:AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQID NO: 1655) and the non-phosphorylated oligonucleotide BsaI-FseI-KpnIrev:CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO: 1656)for introducing the sequencing island B (SI-B) which encodes amino acids GPEPTGPAPSG (SEQ IDNO: 1657) and has the restriction enzyme FseI recognition nucleotide sequence GGCCGGCC inside.
The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested r vector pCWO359 asused in Example 9 to yield 7 containing SI-B. We then generated a library ofXTEN_AM443segments by ining 43 preferred XTEN_AM432 segments from Example 8 and SI-B segmentsfrom pCWO467 at C-terminus using the same zation process described in e 5. This newlibrary of XTEN_AM443 segments was designated LCWO480.
We generated a library ofXTEN_AM13 1 8 segments by recombining segments from libraryLCWO48O ofXTEN_AM443 segments and segments from library LCWO481 _AM875segments using the same dimerization process as in Example 5. This new library ofXTEN_AM13 1 8segment was designated LCWO487.
WO 22617 Example 11: uction ofXTEN_AD864 Using the several consecutive rounds of dimerization, we assembled a collection ofD864 sequences starting from ts ofXTEN_AD36 listed in Example 1. These sequenceswere assembled as described in Example 5. Several isolates from XTEN_AD864 were evaluated andfound to show good expression and excellent lity under physiological conditions. One intermediateconstruct of XTEN_ADS76 was sequenced. This clone was evaluated in a PK experiment in cynomolgusmonkeys and a half-life of about 20 h was measured.
Example 12: Construction ofXTEN_AF864 Using the several consecutive rounds of zation, we assembled a collection ofXTEN_AF864 sequences starting from segments ofXTEN_AF36 listed in Example 3. These sequenceswere assembled as described in Example 5. Several isolates from XTEN_AF864 were ted andfound to show good expression and excellent solubility under physiological conditions. One intermediateuct of XTEN_AF54O was sequenced. This clone was evaluated in a PK ment in cynomolgusmonkeys and a half-life of about 20h was measured. A full length clone ofXTEN_AF864 had excellentsolubility and showed half-life exceeding 60h in cynomolgus s. A second set ofXTEN_AFsequences was assembled including a sequencing island as described in Example 9.
] Example 13: Construction ofXTEN_AG864 Using the several consecutive rounds of dimerization, we assembled a collection ofXTEN_AG864 sequences starting from segments ofXTEN_AD36 listed in Example 1. These sequenceswere assembled as described in Example 5. l es from XTEN_AG864 were evaluated andfound to show good expression and excellent lity under physiological conditions. A full lengthclone of XTEN_AG864 had excellent solubility and showed half-life exceeding 60h in cynomolgusmonkeys.
Example 14: Methods of producing and evaluating CFXTEN with internal and terminalXTEN The design, construction and evaluation of CFXTEN comprising FVIH and one or more XTENis accomplished using a systematic approach. The regions suitable for XTEN insertion sites include, butare to limited to regions at or proximal to the known domain ries of FVHI, exon boundaries,known e loops, regions with a low degree of order, and hydrophilic regions. By analysis of theforegoing, different regions across the sequence of the FVHI B domain deleted (BDD) sequence havebeen identified as ion sites for XTEN, non-limiting examples of which are listed in Tables 5-8, andshown schematically in FIGS. 8 and 9. Initially, individual ucts are created (using methodsdescribed, below) in which DNA encoding a single XTEN or XTEN fragment of a length ranging from 6to 2004 amino acid residues is inserted into the FVHI sequence corresponding to or near (e. g., within 6amino acids) each of the single insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table9, and the resulting constructs are expressed and the red protein then ted for their effects onretention of procoagulant activity using, e. g., one of the in vitro assays of Table 49. For example, usingthe methods described below, constructs are made in which an XTEN sequence is inserted within the AlA2, B, A3, C1 and C2 domain sequences of FVIII, as well as linked to the C-terminus, and the resultingexpressed fusion proteins are evaluated in a chromogenic assay of Table 49, ed to a FVIII notlinked to XTEN. CFXTEN fusion proteins can be r classified acting to high, intermediate and lowcategories based on the activities they exhibit. In those cases where the CFXTEN exhibits activity that iscomparable or modestly reduced compared to FVIII, the insertion site is deemed ble. In thosecases where the activity is intermediate, the insertion site can be adjusted from 1-6 amino acids towardsthe N— or C-terminus of the insertion site and/or the length or net charge of the XTEN may be altered andthe resulting construct(s) luated to determine r the activity is improved. Alternatively, theXTEN is inserted into the construct with flanking cleavage sites; preferably sites that are susceptible tocleavage by proteases found in clotting assays, such that the XTEN is released during the activation ofthe FVIII component, thereby providing additional information about the suitability of the XTENinsertion site in the fusion protein.
Once all of the individual ion sites are evaluated and the favorable insertion sites areidentified, libraries of ucts are created with two, three, four, five or more XTEN inserted in thepermutations of ble sites. The length and net charge of the XTEN (e.g., XTEN of the AE versusAG family) are varied in order to ascertain the s of these variables on FVIII ty andphysicochemical properties of the fusion protein. CFXTEN constructs that retain a desired degree of invitro procoagulant FVIII activity are then ted in vivo using mouse and/or dog models ofhemophilia A, as described in Examples below, or other models known in the art. In addition, constructsare assayed in the presence of FVIII tors and other anti-FVIII dies to determine constructsthat retain activity. In addition, CFXTEN constructs are made that incorporate cleavage sequences at ornear the junction(s) of FVIII and XTEN (e. g., sequences from Table 8) designed to release the XTENand are ted for enhancement of FVIII activity and effects on terminal half-life. By the iterativeprocess of making constructs combining ent insertion sites, varying the length and compositionqualities of the XTEN (e. g., different XTEN families), and evaluation, the skilled artisan obtains, by theforegoing methods, CFXTEN with desired properties, such as but not limited to of procoagulant FVIIIactivity, reduced binding with FVIII inhibitors, enhanced pharmacokinetic properties, ability toadminister to a subject by different routes, and/or enhanced pharmaceutical properties.
Example 15: Methods of producing and evaluating CFXTEN containing FVIII andAE_XTEN] A general scheme for producing and evaluating CFXTEN compositions is presented in ,and forms the basis for the general description of this Example. Using the disclosed methods and thoseknown to one of ordinary skill in the art, together with guidance provided in the illustrative es, ad artesian can create and evaluate CFXTEN fusion proteins comprising XTEN and FVIII orvariants of FVIII known in the art. The Example is, therefore, to be construed as merely illustrative, andnot limitative of the s in any way whatsoever; numerous variations will be apparent to theordinarily skilled artisan. In this Example, a CFXTEN of a factor VIII BDD linked to an XTEN of theAE family of motifs is created.
The general scheme for producing polynucleotides encoding XTEN is presented in FIGS. 11and 12. is a schematic flowchart of representative steps in the assembly of an XTENpolynucleotide construct in one of the embodiments of the invention. Individual oligonucleotides 501 areed into sequence motifs 502 such as a 12-amino acid motif (“12-mer”), which is ligated toadditional sequence motifs from a library that can multimerize to create a pool that encompasses thedesired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI,and KpnI restriction sites 503. The motif libraries include specific sequence XTEN families; e. g., AD,AE, AF, AG, AM, or AQ sequences of Table 3. As illustrated in , the XTEN length, in this case,is 36 amino acid residues, but longer lengths are also achieved by this general process. For example,erization is performed by on, overlap extension, PCR assembly or similar cloning techniquesknown in the art that, in this case, result in a uct with 288 amino acid residues. The resulting poolof ligation ts is gel-purified and the band with the desired length ofXTEN is cut, ing in anisolated XTEN gene with a stopper sequence 505. The XTEN gene can be cloned into a stuffer vector.
In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is thenperformed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting t isthen cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in thegene 500 encoding a CFXTEN fusion protein with a 288 amino acid XTEN linked to the C-terminus ofthe factor VIII. As would be apparent to one of ordinary skill in the art, the methods are applied to createconstructs in alternative rations and with g XTEN lengths or in multiple locations.
] DNA sequences encoding FVIII are conveniently obtained by standard procedures known inthe art from a cDNA library prepared from an riate cellular source, from a genomic library, or maybe created synthetically (e. g., automated nucleic acid synthesis) using DNA ces obtained frompublicly available databases, patents, or literature references. In the present example, a FVIII B domaindeleted (BDD) variant is prepared as described in Example 17. A gene or polynucleotide encoding theFVIII portion of the protein or its complement is then cloned into a construct, such as those describedherein, which can be a plasmid or other vector under control of appropriate ription and ationsequences for high level protein expression in a biological system. A second gene or polynucleotidecoding for the XTEN portion or its ment is cally fused to the nucleotides encoding theterminus of the FVIII gene by cloning it into the construct adjacent and in frame with the gene coding forthe CF, through a ligation or multimerization step. In this manner, a chimeric DNA molecule coding for(or complementary to) the CFXTEN fusion n is generated within the construct. Optionally, a geneencoding for a second XTEN is inserted and ligated in-frame internally to the nucleotides encoding theFVIII-encoding region. The constructs are designed in different configurations to encode variousinsertion sites of the XTEN in the FVIII sequence, including those of Table 5, Table 6, Table 7, Table 8,and Table 9 or those illustrated in FIGS. 8-9. Optionally, this ic DNA molecule is transferred orcloned into r construct that is a more appropriate expression vector; e. g., a vector appropriate for aian host cell such as CHO, BHK and the like. At this point, a host cell capable of expressing thechimeric DNA molecule is transformed with the chimeric DNA molecule, described more completely,below, or by nown methods, depending on the type of ar host, as bed supra.
] Host cells containing the XTEN-FVIH expression vector are cultured in conventional nutrientmedia modified as appropriate for activating the promoter. The culture ions, such as temperature,pH and the like, are those previously used with the host cell selected for expression, and will be ntto the ordinarily skilled artisan. After expression of the fusion protein, culture broth is harvested andseparated from the cell mass and the resulting crude extract retained for purification of the fusion protein.
Gene expression is measured in a sample directly, for example, by conventional Southernblotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA,77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriatelylabeled probe, based on the ces provided herein. Alternatively, gene expression is ed byimmunological of fluorescent methods, such as immunohistochemical staining of cells to quantitately the expression of gene product. Antibodies useful for histochemical ng and/orassay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the dies may be prepared against the FVIII sequence polypeptide using a synthetice based on the sequences provided herein or against exogenous sequence fused to FVHI andencoding a specific antibody epitope. Examples of selectable s are well known to one of skill inthe art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (B-gal) or chloramphenicol transferase (CAT).
] The CFXTEN polypeptide product is purified Via methods known in the art. Procedures suchas gel filtration, affinity ation, salt onation, ion exchange chromatography, size exclusionchromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography orgel electrophoresis are all techniques that may be used in the purification. Specific methods ofpurification are described in Robert K. Scopes, n Purification: Principles and Practice, Charles R.
Castor, ed., Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step ation separations arealso described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr.
A. 679:67-83 (1994).
As illustrated in , the isolated CFXTEN fusion proteins are characterized for theirchemical and activity properties. An isolated fusion protein is characterized, e. g., for ce, purity,apparent molecular weight, solubility and stability using standard methods known in the art. The fusionprotein meeting expected standards is evaluated for actiVity, which can be measured in Vitro or in Vivo bying one of the factor VIII-associated parameters described herein, using one or more assaysdisclosed herein, or using the assays of the Examples or Table 49.
In addition, the CFXTEN FVIII fusion protein is administered to one or more animal species todetermine standard pharmacokinetic parameters and pharmacodynamic properties, as described inExamples 25 and 26.
By the iterative process of producing, expressing, and recovering CFXTEN constructs,followed by their characterization using methods disclosed herein or others known in the art, theCFXTEN compositions comprising CF and an XTEN are produced and evaluated to confirm theexpected properties such as enhanced lity, enhanced stability, improved pharmacokinetics andreduced immunogenicity, leading to an overall enhanced therapeutic activity compared to theponding unfused FVIII. For those fusion proteins not possessing the desired properties, a differentsequence or configuration is constructed, expressed, isolated and evaluated by these methods in order toobtain a composition with such properties.
Example 16: Construction of expression plasmids for BDD FVIII] I. uction of B domain deleted FVIII (BDD FVIII) expression vectors The expression vector encoding BDD FVIII was created by cloning the BDD FVIII openreading frame into the pcDNA4 vector (Invitrogen, CA) containing a polyA to allow for optimalmammalian expression of the FVIII gene, resulting in a construct designated pBC0100. Several naturalsites were identified within this construct for cloning use, including BsiWI 48, AflII 381, PshAI 1098,KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI4500, PmeI 4527. To facilitate assay development, nucleotides encoding Myc and His tag wereintroduced into the FVIII open reading frame. pBC0100 was PCR amplified using the followingprimers: 1) F8-BsiWI-F: tattccCGTACchcgccaccATGCAAATAGAGCTCTCCACCT (SEQ ID NO:1658); 2) F8-nostop-XhoI-R1: GGTGACCTCGAGcgtagaggtcctgtgcctcg (SEQ ID NO: 1659) tointroduce BsiWI and XhoI in appropriate locations. The PCR product was digested with BsiWI andXhoI. PcDNA4-Myc-His/C was digested with Acc651 and XhoI, which generated two ts of 5003and 68 bps. The 5003bps product was ligated with the digested PCR’ed FVIII fragment and used forDH5alpha transformation. The enzymes Acc65I and BsiWI create compatible ends but this ligationdestroys the site for future ion. The ing construct was ated pBC0102 (pcDNA4-FVIII_3-Myc-His). To facilitate the design and execution of future cloning strategies, especially onesinvolving the creation of BDD FVIII expression constructs that contain multiple XTEN insertions, weed additional unique restriction enzyme sites to orate, including BsiWI 908, NheI 1829 andClaI 3281. The introduction of these sites was done via the QuikChange method (Agilent, CA)individually. The resulting construct was designated pBC0112 (pcDNA4-FVIII_4-Myc-His). To avoidproblems that may arise from the linker peptides that connects between Myc/His and FVIII/Myc, and toremove restriction enzyme sites that are preferred for future XTEN ion, we mutated the sequencesencoding the peptide sequences from ARGHPF (SEQ ID NO: 1660) to AETA (SEQ ID NO:178) (between FVIII and Myc), NMHTG (SEQ ID NO: 1661) to SPATG (SEQ ID NO: 1662) (betweenMyc and His) via the QuikChange method. The construct was ated pBC0114 (pcDNA4-FVIII_4-AETA—Myc-SPATG-His ('GAGSPGAETA' and ' disclosed as SEQ ID NOS 178 and1662, respectively)) (sequence in Table 21), which was used as the base vector for the design andcreation of other expression vectors incorporating XTEN sequences. Expression and FVIII activity datafor this construct are ted in II. Construction of B domain deleted FVIII (BDD FVIII) expression vectorsWO 22617 The gene encoding BDD FVIII is synthesized by GeneArts (Regensburg, Germany) in thecloning vector pMK (pMK-BDD FVIII). The BDD FVIII proteins contain 1457 amino acids at a totalmolecular weight of 167539.66. There are 6 domains within the wild-type FVIII protein, the A1, A2, B,A3, C1 and C2 s. In the BDD FVIII protein, most of the B domain has been deleted as it wasshown to be an ctured domain and the removal of the domain does not alter critical functions ofthis protein. The pMK vector used by GeneArts contains no promoter, and can not be used as anexpression vector. Restriction enzyme sites NheI on the 5’ end and SfiI, SalI and XhoI on the 3’ end areintroduced to facilitate subcloning of the DNA sequence encoding BDD FVIII into expression s,such as 9-HS (Millipore). Several unique restriction enzyme sites are also introduced into theFVIII ce to allow further manipulation (e. g., insertion, mutagenesis) of the DNA sequences.
Unique sites listed with their cut site e, but are not limited to: SacI 391, AfiII 700, SpeI 966, PshAI1417, Acc6512192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, ApaI 3893,Bsp1201 3893, SwaI 4265, OliI 4626, XbaI 4644, and BstBI 4673. . The HindIII site s at the veryend of the A2 domain and can potentially be used for modification of the B domain. The synthesizedpMK-BDD FVIII from GeneArts does not contain a stop codon. The stop codon is introduced byamplifying a 127 bp fragment of FVIII using the following primers: 5’-GTGAACTCTCTAGACCCACCG-3’ (SEQ ID NO: 1663); 5’-CTCCTCGAGGTCGACTCAGTAGAGGTCCTGTGCCTCG-3’ (SEQ ID NO: 1664). The fragment isdigested with XbaI and SalI, and ligated to XbaI /SalI digested pMK-BDD FVIII. The ligated DNAmixture is used to transform DH5a bacterial cells. Transformants are ed by DNA miniprep and thedesired constructs are confirmed by DNA sequencing. The uct named pBCOO27 (pMK-BDDFVIII-STOP) contains coding sequences that encode the BDD FVIII protein. The pBC0027 construct isthen ed with NheI /SalI, and d with NheI/SalI ed CET1019-HS vector (Millipore). TheCET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate geneexpression. The ligated DNA mixture is used to transform DH5a bacterial cells. ormants arescreened by DNA ep and the desired constructs are confirmed by DNA sequencing. The finalconstruct is designated pBCOO25 (CET1019-HS-BDD FVIII-STOP), which encodes the BDD FVIIIprotein under the control of a human CMV promoter. Introduction of the pBCOO25 construct intomammalian cells is expected to allow sion of the BDD FVIII protein with procoagulant activity.
Example 17: Construction of expression plasmids for BDD FVIII Containing XTEN 1. B domain AE42 Insertion Two PCR reactions were run in parallel to insert XTEN_AE42 into the remaining B domainregion of the BDD FVIII constructs. The PCR reactions involved the following primers:cgaaagcgctacgcctgagaGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCTGAAACCCCTGCCTCGAGCccaccagtcttgaaacgcc (SEQ ID NO: 1665);TGATATGGTATCATCATAATCGATTTCCTCTTGATCTGACTG (SEQ ID NO: 1666);agcttgaggatccagagttc (SEQ ID NO: 1667);tctcaggcgtagcgctttchTTGTCCCCTCTTCTGTTGAGGTGGGGGAGCCAGCAGGAGAACCTGGCGCGCCgttttgagagaagcttcttggt (SEQ ID NO: 1668). The PCR products then served as templates, and asecond PCR was performed to introduce the XTEN_AE42 into the FVIII encoding tide sequencesflanked by BamHI and ClaI. This PCR product was digested with BamHI and ClaI simultaneously withthe digestion of PBCOl 14 with the same two enzymes. The PCR product was ligated to the digestedvector. This construct was ated pBC0135 4-FVIII_4XTEN_AE42-GAGSPGAETA—Myc-SPATG-His) ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, tively),and encodes the BDD FVIII with an AE42 XTEN incorporated within the residual B-domain. 2. AE42 Insertion and R1648A mutation The QuikChange method (Agilent, CA) was employed to introduce an R1648A mutation intoPBC0135. This construct was designated pBC0149 (pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA-Myc-SPATG-His_R1648A) ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662,respectively), eliminating that FVIII processing site. 3. B domain AE288 ion XTEN_AE288 was PCR amplified using the following primers:tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1669) andtggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1670). PBCOO75 was used as the template for thisPCR reaction. The PCR product was digested with AscI and XhoI, and PBC0135 was ed with thesame enzymes. The PCR product was ligated to the PBC0135 fragment. This construct was designatedpBC0136 (pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA—Myc-SPATG-His) ('GAGSPGAETA' and'SPATG' disclosed as SEQ ID NOS 178 and 1662, tively), and encodes the BDD FVIII with anAE288 XTEN incorporated within the residual B-domain. 4. AE288 Insertion and R1648A mutation XTEN_AE288 was PCR ied using the following primers:tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1671) andtggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1672). Construct pBCOO75 was used as the templatefor this PCR reaction. The PCR product was digested with AscI and XhoI, and pBC0149 was digestedwith the same enzymes. The PCR product was ligated to the pBC0149 fragment. This construct wasdesignated pBC0137 (pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA—Myc-SPATG-His R1648A)PGAETA' and ' disclosed as SEQ ID NOS 178 and 1662, tively) and contains anAE288 XTEN sequence internal to the B domain, with the R1648A mutation eliminating that FVIIIprocessing site. 3. B domain AE144 AG144 AG288 Insertions with and without R1648A mutations Select XTEN nts were PCR ied to introduce AscI and XhoI sites to the 5’ and 3’end respectively. The PCR product was digested with AscI and XhoI, and pBC0135 (for R1648) orpBC0149 (for A1648) were digested with the same enzymes. The PCR product was d to thepBC0135 or pBC0149 vector. These constructs were designated pSDOOOS, 6, 7, 8, 17 and 18.
Construction of expression plasmids for BDD FVIII with XTEN insertion at the C terminus 1. C terminal AE288 insertion XTEN_AE288 was PCR amplified using the following primers:ggggccgaaacggccggtacctcagagtctgctacc (SEQ ID NO: 1673) and tgttcggccgtttcggcccctggcgcactgccttc(SEQ ID NO: 1674). The construct pBC0075 was used as the te for this PCR reaction. The PCRproduct was digested with SfiI, and pBC0114 was digested with the same enzyme. The PCR productwas d to the digested pBC0114 fragment. This construct was designated pBC0145 4-4-XTEN_AE288-GAGSPGAETA—Myc-SPATG-His) ('GAGSPGAETA' and 'SPATG' disclosedas SEQ ID NOS 178 and 1662, respectively), and encodes an AE288 sequence at the C-terminus of theBDD FVIII.] 2. C terminal AG288 insertion XTEN _AG288 was designed and synthesized by DNA2.0 (Menlo Park, CA). The synthesizedgene was PCR amplified using the ing primers: ggggccgaaacggccccgggagcgtcacc (SEQ ID NO:1675) and tgttcggccgtttcggcccctgacccggttgcccc (SEQ ID NO: 1676). The PCR product was digestedwith SfiI, and PBC0114 based vector was digested with the same enzyme. The PCR product was ligatedto the digested PBC0114 fragment. This construct was designated pBC0146 4-FVIII_4-XTEN_AG288-GAGSPGAETA—Myc-SPATG-His) ('GAGSPGAETA' and 'SPATG' disclosed as SEQID NOS 178 and 1662, respectively), and encodes an AG288 ce at the C-terminus of the BDDFVIII. 3. C terminal AE/AG144 288 864 insertionsAscI and XhoI sites were introduced into the 4 based vector via QuikChange methods using theprimers: 5037-PBC0114-AscI-XhoI-F:CAGGACCTCTACGGCGCgccagcctcgaGCGAACAAAAACTCATCTCAGAAGAGG (SEQ ID NO:1677); 503 8-PBC01 14-AscI-XhoI-R:CCTCTTCTGAGATGAGTTTTTGTTCGthgaggctgchCGCCGTAGAGGTCCTG (SEQ ID NO:1678). Various XTEN fragments were PCR amplified with AscI and XhoI introduced into the 5’ and 3’end respectively. The PCR product was ligated to the digested PBC0114 vector. These constructs weredesignated pSD0013, 4, pSD0015, pSD0016, pSD0019 and pSD0020.
Construction of expression plasmids for BDD FVIII with inter- and intra- domain XTENinsertions 1. AE7 AE42 and AEl44 Insertions Four distinct strategies are used for insertion of AE42 into the designated sites (e.g., the naturalor introduced restriction sites BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094,ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI 4527, BsiWI 908, NheI1829 and ClaI 3281) within the BDD FVIII encoding sequence, each contributing to the creation ofseveral constructs. By design, these insertions of AE42 create AscI and XhoI sites flanked on either sideof the insertion allowing for uction/substitution of longer XTENs, as well as XTEN with differentsequences or incorporated cleavage ces, as needed. Specifically, the constructs that containXTEN_144 insertions are listed in Table 21. These insertions were created by replacing either AE7 orAE42 with a PCRed XTEN_144 fragment flanked by AscI and XhoI sites. 2012/046326 2. Double PCR-mediated method Two PCR reactions are run in parallel to insert XTEN_AE42 into the ated site. The twoPCR reactions introduce XTEN on either the 3’ or the 5’ end via use of a long primer that contains lXTEN. The PCR products then serve as templates, and a second PCR is performed to uce theXTEN_AE42 into the FVIH encoding nucleotide sequences flanked by select ction enzyme sites.
This PCR product is digested with the appropriate enzymes simultaneously with the digestion ofPBC0114 using the same two enzymes. The PCR product is ligated to the ed vector. Using this, ucts are created designated pBC0126, pBC0127, pBC0128, and pBC0129, resulting inAE42 insertions at the R3, R3, P130, L216 locations respectively. The sequences are listed in Table 21.
Select XTEN_l44 sequences can then be PCRed to introduce Ascl and Xhol sites on either end of thefragment, and ligate to digested FVHI-XTEN_AE42 construct. For instance, pSD0053 was created byreplacing the AE42 of pBC0129 with XTEN_AE144. Other XTEN_l44 constructs were created via thesame strategy and are listed in Table 21. 3. QuikChange mediated two step cloning method The QuikChange method is employed to uce XTEN_AE7 encoding sequences that areflanked by Ascl and Xhol into designated sites. The resulting intermediate construct is then digestedwith Ascl and Xhol. E42 or XTEN_AE144 is PCR amplified to introduce the two sites anddigested accordingly. The vector and insert are then ligated to create the final constructs. The sequencesare listed in Table 21. 4. Three PCR type II ction enzyme mediated ligation method Three PCR reactions are performed to create two pieces of FVIII encoding fragments flankedby one type I restriction enzyme that correlates with a unique site within the FVHI_4 gene and one typeII enzyme (e. g. Bsal, Bbsl, Bqul), the third PCR reaction created the XTEN_AE42 flanked by two typeII restriction enzyme sites. The three PCR fragments are digested with appropriate enzymes and ligatedinto one linear piece that contains the XTEN_AE42 insertion within a fragment of FVIII encodingsequences. This product is then digested with appropriate unique enzymes within the FVIII encodingces and ligated to the 4 construct ed with the same enzymes, and result in constructsdesignated pBC0130 (with XTEN insertion at residue P333), pBC0132 (with XTEN insertion at residueD403), pBC0133 (with XTEN insertion at residue R490). The sequences are listed in Table 21. SelectXTEN_l44 ces can then be PCRed to introduce Ascl and Xhol sites on either end of the fragment,and ligate to digested FVIH-XTEN_AE42 construct. For instance, pSD0001 and pSD0003 were createdby replacing the AE42 of pBC0132 with XTEN_AE144 and XTEN_AG144 respectively. OtherXTEN_l44 constructs listed in Table 21 were created via the same strategy. 5. Custom gene sis Custom gene synthesis is med by GeneArt (Regensburg, Germany). The genes areed so that they include nucleotides encoding the XTEN_AE42 inserted in the designated site(s)and the genes are flanked by two unique restriction enzyme sites selected within the FVHI_4 gene. Thesynthesized genes and PBC0114 are digested with appropriate enzymes and ligated to create the finalproduct with the BDD FVIII incorporating the XTEN_AE42 between the restriction sites. SelectXTEN_144 sequences can then be PCRed to introduce AscI and Xhol sites on either end of the fragment,and ligate to digested FVIII-XTEN_AE42 construct.
Construction of expression plasmids with dual XTEN insertions in the B domain and at the Cterminus The construct pBC0136, which encodes the BDD FVIII with an AE288 XTEN incorporatedwithin the residual B-domain, is digested with BamHI and ClaI, and the resulting 1372bps fragment fromthis digestion is the insert. The construct pBC0146 is digested with BamHI and ClaI, and the 9791bpspiece from this digestion is the vector. The vector and insert are ligated together to create pBC0209,containing an AE288 insertion within the B domain and an AG288 on the C terminus. The same strategyis utilized to create constructs containing two AE288 insertions in the B domain and at the C terminus,respectively, using PBC0145 as the vector.
Construction of expression plasmids with le XTEN insertions The construct pBC0127, which encodes an AE42 XTEN at the R3 position of FVHI, is edwith BsiWI and Aflll, and the resulting 468bps fragment from this digestion is the . The uctpBC0209 is digested with BsiWI and AflH, the 10830bps piece from this digestion is the vector. Thevector and insert are ligated together to create a uct designated pBC0210, containing an AE42insertion in the A1 , an extra three ATR amino acid to restore the signal cleavage sequence, anAE288 XTEN insertion within the B domain and an AG288 on the C terminus. The same methodologyis used to create ucts encoding multiple XTEN at the natural and introduced restriction sites; e. g.,BsiW148, AflII 381, PshAl 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, Xba14325, NotI4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI 4527, BsiWI 908, NheI 1829 and Clal 3281. uction of BDD FVHI-Internal-XTEN_AE288 expression vectors Two BsaI restriction enzyme sites are introduced into the PBC0027 pMK-BDD FVIII constructbetween the base pair 2673 and 2674 using the QuikChange method following manufacturer’s ol(Agilent Technologies, CA). The inserted DNA sequences are gggtctcccgcgccagggtctccc, and theresulting construct is ated pBC0205 (sequence in Table 21). The DNA ce encoding AE288(or other variants and lengths of XTEN; e. g. AE42, AG42, AG288, AM288) is then PCR’ed with primersthat introduce BsaI sites on both the 5’ and 3’. The pBC0205 vector and the insert 288) are thendigested with BsaI and ligated to create pBC0206, which encodes the FVIII gene with an XTEN_AE288insertion within the B domain (sequence in Table 21). The pBC0206 construct is then digested withNheI /SalI, and ligated with NheI/Sall digested CET1019-HS vector (Millipore). The 9-HSvector contains a human CMV promoter and a UCOE ce to facilitate gene expression. The ligatedDNA e is used to transform DH5a bacterial cells. ormants are screened by DNA miniprepand the desired constructs are confirmed by DNA sequencing. The final construct is designatedpBC0207 (CET1019-HS-BDD FVHI-STOP), which s the BDD FVIII protein under the control ofa human CMV promoter (sequence in Table 21). Introduction of the pBC0207 construct into mammaliancells is expected to allow expression of the BDD FVIII protein with an internal XTEN_AE288. Thesame protocol is used to introduce, transform and express constructs containing other variants andlengths of XTEN; e. g. AE42, AG42, AG288, AM288, AE864, AG864, or other XTEN of Table 4.
Construction of BDD FVIII-/—XTEN_AE864 expression vectors The BDD FVIII fragment with NheI and SfiI flanking the 5’ and 3’ end is generated bydigesting the pBC0025 construct. This digested fragment is then ligated to a NheI/SfiI digested pSecTagvector (pBCOO48 pSecTag-FVIII-/—XTEN_AE864) encoding the FVIII followed by the XTEN_AE864sequence. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants arescreened by DNA miniprep and the desired ucts are confirmed by DNA sequencing. The finalconstruct is 0, which encodes the BDD FVIII-/—XTEN_AE864 protein under the control of ahuman CMV promoter. Introduction of the pBCOO6O construct into mammalian cells is expected tos the FVIII protein with a C terminal XTEN fusion (BDD FVIII-/—XTEN_AE864) withprocoagulant activity. uction of BDD /FXI/—XTEN_AE864 expression s The BDD FVIII fragment with NheI and SfiI flanking the 5’ and 3’ end is generated bydigesting the pBC0025 construct. This digested fragment is then ligated to a NheI/SfiI digested pSecTagvector (pBCOO47 pSecTag-FVIII-/FXI/—XTEN_AE864) encoding the FVIII followed by the FXIcleavage sequence (/FXI/) and XTEN_AE864. The ligated DNA mixture is used to transform DHSabacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmedby DNA sequencing. The final construct is pBCOOS l, which encodes the BDD FVIII-/FXI/—XTEN_AE864 protein under the control of a human CMV promoter. Introduction of the pBCOOSlconstruct into mammalian cells is expected to s the FVIII protein with a C terminal XTEN fusion(BDD FVIII-/FXI/—XTEN_AE864), which could be subsequently cleaved by FXI, therefore liberating theBDD FVIII protein with procoagulant activity.
Construction of BDD FVIII-/FXI/—XTEN expression vectors comprising AE288 or AG288 The fused AE864 XTEN sequence in pBCOO6O is replaced by digesting the XTEN cesAE288 and AG288 with BsaI and HindIII. A subsequent ligation step using the respective AE288 orAG288 XTEN fragment and indIII digested pBCOOS 1 allows the ge of the AE288 orAG288 sequences into the BDD FVIII expression . The resulting final ucts are pBCOO6l forBDD FVIII-AE288 and pBCOO62 for BDD FVIII-AG288. Introduction of the pBCOO6l construct intomammalian cells is ed to express the FVIII protein with a C-terminal AE288 XTEN fusion (BDDFVIII-/—XTEN_AE288) with procoagulant ty. Introduction of the pBCOO62 construct intomammalian cells is expected to express the FVIII protein with a C-terminal AG288 XTEN fusion (BDDFVIII-/—XTEN_AG288) with procoagulant activity.
Construction of BDD FVIII-/FXI/—XTEN sion vectors with alternate XTEN] The fused XTEN sequence in pBCOOS l is replaced by digesting DNA encoding other XTENsequences (e. g. other variants and lengths of XTEN; e.g. AE42, AG42, AG288, AM288) with BsaI andHindIII. A ligation using the XTEN nt and indIII digested pBCOOS 1 allows the exchangeof the various XTEN-encoding sequences into the BDD FVIII expression , providing the alternateconstructs. Introduction of the alternate constructs into mammalian cells is expected to express the FVIIIprotein with a C-terminal XTEN (BDD /FXI/—XTEN) that can be subsequently cleaved by FXI,releasing the FVIII, resulting in procoagulant FVIII fusion with procoagulant activity. e 18: uction of expression ds for FVIII signal peptide-XTEN-/FXI/—BDD FVIII Construction of expression vectors for FVIII signal peptide-XTEN_AE864 The coding sequences for the FVIII signal peptide is generated by annealing the following twooligos: 5’-CTAGCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGGGTCTCC-3’ (SEQ ID NO: 1679); 5’-ACCTGGAGACCCACTAAAGCAGAATCGCAAAAGGCACAGAAAGAAGCAGGTGGAGAGCTCTATTTGCATG-3’ (SEQ ID NO: 1680). The annealed oligos are flanked by the NheI and BsaIrestriction enzyme sites on either end, and is ligated to saI digested pCWO645 vector whichencodes the FVII-XTEN_AE864. The ligated DNA mixture is used to transform DH5a bacterial cells.
Transformants is screened by DNA miniprep and the desired constructs are confirmed by DNAsequencing. The final construct is designated pBC0029, which encodes the signal peptide-XTEN_AE864 protein under the l of a human CMV promoter. This construct is used as anintermediate construct for ng an expression construct with XTEN fused on the N-terminus of theFVIII protein, and can also be used as a master plasmid for creating expression constructs that allowXTEN fusion on the N-terminus of a secreted protein.
Construction of signal peptide-XTEN_AE864-/FXI/—BDD FVIII expression vectors An l800bp fragment within the FVIII coding region is amplified using s that uceNheI-BbsI-/FXI/—AgeI sites on the 5’ and endogenous KpnI restriction enzyme on the 3’ end. TheNheI/KpnI digested FVIII fragment is ligated with NheI/KpnI digested pBC0027 vector. The ligatedDNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprepand the desired constructs are confirmed by DNA sequencing. The resulting construct is designatedpBC0052, which contains sequences that encode the /FXI/—FVIII protein without the FVIII signalpeptide. This uct is used as an intermediate construct for ng an expression construct withXTEN fused on the N-terminus of the FVIII protein.
The pBC0052 vector is digested with BbsI/XhoI s, and is used to ligate with Bbsi/XhoIdigested pBC0029. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformantsare screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The finalconstruct is designated pBCOOS3, which encodes the signal peptide-XTEN_AE864-/FXI/—BDD FVIIIprotein under the control of a human CMV er. Introduction of the pBCOOS3 construct intomammalian cells is expected to express the FVIII protein with an N-terminal XTEN fiJsion (signalpeptide-XTEN_AE864-/FXI/—BDD FVIII), which could be subsequently cleaved by FXI, oreliberating the BDD FVIII protein.
Construction of signal peptide-XTEN —BDD FVIII expression vectors The fused XTEN sequence in pBC0053 can be replaced by digesting other XTEN fragments(e. g. AM, AF, AG) with Bsal and Bbsl. A ligation using the XTEN fragment and BsaI/Bbsl digestedpBC0053 allows the exchange of various XTEN pieces (e. g. AM, AF, AG) into the BDD FVIIIexpression vector. Various XTEN fusions can increase the half lives of these proteins differently,allowing modification of the properties (e. g. efficacy, potency) of these proteins. Introduction of any ofthese fusion constructs into mammalian cells is expected to express the FVIII protein with an inalXTEN fusion (signal peptide-XTEN-/FXl/-BDD FVHI), in which the fused XTEN peptide can besubsequently cleaved by FXI, generating the BDD FVIII protein.
Example 19: Construction of BDD FVIII with interdomain XTEN insertion Construction of BDD FVIII expression vectors with an XTEN insertion at the A2-B domainboundaries The pBC0027 construct (pMK—BDD FVIll-STOP) is a cloning vector designed to contain theBDD FVIH protein coding sequences, but not a promoter positioned to initiate the sion of BDDFVHI. This uct is used for manipulation of the coding sequences of BDD FVIII as the vectorbackbone contains very few restriction enzyme sites. therefore allowing easy g strategies. TheBDD FVIH proteins n 1457 amino acids at a total molecular weight of .66. There are 6domains within the wild-type FVIII protein, the Al, A2, B, A3, C1 and C2 domains. In the BDD FVIIIprotein, most of the B domain has been d as it is believed to be an unstructured domain and theremoval of the domain does not alter critical ons of this protein. However, the B domainboundaries seem to be ent positions for creating XTEN fusions to allow extension of the proteinhalf lives.
Within the pBC0027 construct, there is a unique Hindlll restriction enzyme site at the boundaryof A2-B junction. The XTEN (e.g., sequences of Tables 4, or 13-17) are amplified using primers thatintroduce a Hindlll and FXI cleavage site on either end of the XTEN coding sequence. The fused XTENsequence can be altered by amplifying various XTEN fragments. Various XTEN fusions can increasethe half lives of these proteins ently, allowing ation of the properties (e.g. efficacy, potency)of these ns. The l-/FXl/-XTEN-/FXl/-Hindlll fragment is digested with l and ligatedwith l digested pBC0027. The ligated DNA mixture is used to transform DH5a bacterial cells.
Transformants are screened by DNA miniprep and the desired constructs are ed by DNAsequencing. The final construct is designated pBC0054, which encodes the BDD FVIII protein with aninterdomain XTEN fusion (FVlll(Al -A2)-/FXl/-XTEN-/FXl/-FVHI(Cl-C2)) but not a promoter toinitiate gene expression.
The pBC0054 construct is digested with Nhel /Sall, and ligated with Nhel/Sall digestedCETlOl9-HS vector (Millipore). The 9-HS vector contains a human CMV promoter and aUCOE sequence to facilitate gene expression. The d DNA mixture is used to transform DH5abacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmedby DNA sequencing. The final construct is designated pBCOOSS (CETlOl9-HS- FVIII(Al-A2)-/FXl/-XTEN-/FXl/-FVHI(Cl-C2)), which encodes the BDD FVIII protein with an interdomain (inter-A2/BWO 22617 domain) XTEN fusion (FVIII(A1-A2)-/FXI/—XTEN-/FXI/—FVIII(C1-C2)) under the control of a humanCMV promoter. Introduction of the pBC0055 construct into mammalian cells is ed to express theBDD FVIII protein with an interdomain XTEN fusion (A1 -A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)), which could be subsequently cleaved by FXI, therefore ting the BDD FVIII n.
Construction of BDD FVIII sion vectors with an XTEN insertion at the A1-A2 domainboundaries The pBC0027 construct is designed as a template for two PCR reactions using the ingfour s:(Reaction I) 5’-ATGATGGCATGGAAGCCTAT-3’ (SEQ ID NO: 1681); 5’-ATCCCTCACCTTCGCCAGAACCTTCAGAACCCTCACCTTCAGAACCTTCACCAGAACCTTCACCATCTTCCGCTTCTTCATTATTTTTCAT-3’ (SEQ ID NO: 1682).
(Reaction II) 5’-TTCTGGCGAAGGTGAGGGATCTGAAGGCGGTTCTGAAGGTGAAGGTGGCTCTGAGGGTTCCGATGATGATCTTACTGATTCTGAAAT-3’ (SEQ ID NO: 1683); 5 ’-TATTCTCTGTGAGGTACCAGC-3’ (SEQ ID NO: 1684).
The PCR products generated are 150bps and 800 bps respectively. The 800 bp product is usedas the template for the next round of PCR reaction with the 150bp t as one primer and 5 ’-TATTCTCTGTGAGGTACCAGC-3’ (SEQ ID NO: 1685) as the other. The product for the secondround of PCR is 930 bps and is digested with PshAI and ACC651 restriction enzymes. ThisPshAI/Acc651 flanked DNA fragment is ligated with PshAI/Acc651 digested pBC0027. The ligatedDNA mixture is used to transform DH5a bacterial cells. Transformants is screened by DNA miniprepand the desired constructs are confirmed by DNA sequencing. The final construct is designatedpBC0058 (pMK-BDD FVIII-D345-XTEN_Y36), which encodes the BDD FVIII protein with aninterdomain (inter-A1/A2 domain) XTEN fusion after the D345 residue.
] The pBC0058 construct is digested with NheI /SalI, and ligated with NheI/SalI digestedCET1019-HS vector pore). The CETlOl9-HS vector contains a human CMV promoter and aUCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5abacterial cells. Transformants are screened by DNA miniprep and the desired ucts are confirmedby DNA cing. The final construct is designated pBC0059 (CET1019-HS-BDD FVIII D345-XTEN_Y36), which encodes the BDD FVIII protein with an interdomain (inter-A1/A2 domain) XTENfusion after the D345 residue under the control of a human CMV promoter. Introduction of the pBC0059construct into mammalian cells is expected to express the BDD FVIII protein with an interdomain XTENfusion (BDD FVIII D345-XTEN_Y36).
Example 20: uction of FVIII with intradomain XTEN insertion Construction of BDD FVIII expression vectors with an XTEN insertion after P598 (within theA2 domain) The coding sequences for XTEN_Y36 is amplified using PCR techniques with the followingprimers: 5 ’- PCT/U82012/046326GAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGGTGAAGGTTCTGGTGAAGG-3’ (SEQ ID NO: 1686)’-AACTCTGGATCCTCAAGCTGCACTCCAGCTTCGGAACCCTCAGAGCC-3’ (SEQ ID NO:1 687).
The 184 bp PCR product is flanked by the KpnI and BamHI restriction enzyme sites on eitherend, and is ligated to KpnII/BamHI digested pBC0027 vector which encodes the BDD FVIII gene. Theligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNAminiprep and the d constructs are med by DNA sequencing. The final construct is designatedpBC0056, which contains DNA sequences ng the FVIII protein with an XTEN_Y36 fusion afterthe P598 e. This cloning strategy is used to introduce various forms ofXTEN into the BDD FVIIIprotein by altering the template for the PCR reaction and changing the primers ingly.
The pBC0056 construct is digested with NheI /SalI, and ligated with NheI/SalI digestedCET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and aUCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5abacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmedby DNA sequencing. The final construct is designated pBC0057 (CET1019-HS- FVIII P598-XTEN_Y32), which s the BDD FVIII protein with an intradomain (within A2 ) XTENfusion under the control of a human CMV promoter. Introduction of the pBC0057 construct intomammalian cells is expected to express the BDD FVIII protein with an intradomain XTEN fusion (FVIIIP598-XTEN_Y32).
Construction of BDD FVIII sion vectors with other omain XTEN insertions To introduce various XTEN segments into other intradomain sites within BDD FVIII (e. g., theXTEN of Tables 4, or 13-17), primers are designed that amplify XTEN with an overhang that can annealwith BDD FVIII. The coding sequence of FVIII (pMK-BDD FVIII) is designed with various uniquerestriction enzyme sites to allow these ic insertions. The unique restriction enzymes are listedbelow with their cut site: NheI 376, SacI 391, AfiII 700, SpeI 966, PshAI 1417, 2192, KpnI2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, ApaI 3893, Bsp1201 3893, SwaI 4265, CHI4626, XbaI 4644, BstBI 4673, SalI4756, and XhoI 4762. The NheI and SalI sites on either end of thecoding sequence are used to insert the DNA fragment into a human CMV promoter driven vector, theCET1019-HS (Millipore) for expression in mammalian cells. These constructs express the BDD FVIIIprotein with an XTEN fusion with sequences listed in Table 21.
Example 21: Construction of FVIII with XTEN insertions CFXTEN with two XTEN: To obtain CFXTEN with two XTEN insertions in various regions (from ini to C-termini: A1 -R1, A1-R2, A2-R1, A2-R2, B domain, a3, A3-R1, A3-R2, C-termini), cts thatexpressed fusions with single-XTEN insertions that retained FVIII ty were utilized. The codingsequence of FVIII (pBC0114 pcDNA4-FVIII_4-X10-Myc-SPATG-His extra RE) ('SPATG' disclosed asSEQ ID NO: 1662) was designed with various unique restriction enzyme sites to allow these specificcombinations. The unique restriction enzymes are listed in Table 18 below with their relative sitesbetween different regions: BsiWI en N-termini and A1 -R1), AflII (between A1 -R1 and A1-R2),Nhel (between A1-R2 and , KpnI (between A2-R1 and A2-R2), BamHl (between A2-R2 and Bdomain), Clal (between a3 and A3-R1), PflMI (between A3 -R1 and A3-R2), Xbal (between A3-R2 andC-termini), Agel (between FVIII C-termini and stop codon). Building blocks and restriction enzymes forcloning the ies were chosen, as listed in the table below. The chosen components in each regionwere mixed at molar ratio of 1 :1, and two sets of DNA mixtures were digested with unique restrictionenzymes. DNA fragments were separated with 1% agarose gel and purified by Qiagen gel extraction kit.
DNA with XTEN insertion in the first desired region was regarded as the insert (the smaller DNAnt in agarose gel), while DNA with XTEN insertion in the second d region was regarded asvector (the bigger DNA fragment in agarose gel). The insert and vector were ligated in order toreconstitute the plasmid. The ligated DNA e was used to transform DH50L E. coli ent hostcells. Transformants were screened by rolling circle amplification (RCA) and Sanger sequencing to coverimately 3-4 times the potential library size. Unique clones were fied and minipreped. Twodistinct restriction digestions were then used to further confirm the integrity ofXTEN in each region. Theamino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed inTable 21.
CFXTEN with one or two XTEN insertions within the B/a3 domain and C terminus: The B/a3 domain and inus of FVIII are unstructured regions that tolerated XTENinsertions well. The B/a3 domain further mediated interactions with other cofactors, including the vonWillibrand Factor. To investigate the optimal XTEN insertions at the B/a3 domain, select deletions andmutations of the region were made via PCR-based mutagenesis methods. Select PCR reactions and thevectors were digested with unique restriction enzymes as listed in Table 18. DNA fragments wereted with 1% agarose gel and purified by Qiagen gel extraction kit. DNA with XTEN ion inthe first d region was regarded as the insert (the smaller DNA fragment in agarose gel), while DNAwith XTEN insertion in the second desired region was regarded as vector (the bigger DNA fragment inagarose gel). The insert and vector were ligated in order to reconstitute the plasmid. The ligated DNAmixture was used to transform DH50t E. coli competent host cells. Transformants were ed bycolony PCR and Sanger sequencing to cover approximately 8X the potential library size. Unique cloneswere identified and minipreped. One three-enzyme restriction digestion was then used to further confirmthe integrity ofXTEN in each region. The amino acid and the encoding DNA sequences for the resultingCFXTEN fusion proteins are listed in Table 21.
Table 18. Clonin desi n for FVIII libraries with two XTEN ionspSD0005, pSD0006, pSD0007, pSD0008,LSD0001 pSD0017, pSD0018, pBC0136, pBC0137 pSD0013 (C-termini) NheI -- ClaI(B-domain)pSD0005, pSD0006, pSD0007, pSD0008,LSD0002 pSD0017, pSD0018, pBC013 6, pBC013 7 pSD0014 (C-termini) NheI -- ClaI(B-domain)W0 2013/122617 PCT/U82012/046326Library . Vector components RestrictionInsert components (XTEN region)ID QETEN re ion; en mespSD0005, pSD0006, pSD0007, pSD0008,LSD0003 pSD0017,pSD0018,pBC0136,pBC0137 pSD0019 (C-termini) NheI--C1aI(B-domain)pSD0005, pSD0006, pSD0007, pSD0008,LSD0004 pSD0017,pSD0018,pBC0136,pBC0137 pSD0020 (C-termini) NheI--C1aIain)pSD0045,pSD0046,pSD0048,pSD0049,LSD0005 pSD0001(A2-R1) Bs1WI AflII. __pSD0050,pSD0051,pSD0052 (A1-R1),pSD0046,pSD0048,pSD0049,LSD0006 2(A2-R1) Bs1WI AflII. __pSD0050,pSD0051,pSD0052 (A1-R1)pSD0045,pSD0046,pSD0048,pSD0049,LSD0007 pSD0003 (A2 R1) ._ Bs1WI AflII0,pSD0051,pSD0052 (A1-R1)pSD0045,pSD0046,pSD0048,pSD0049,LSD0008 pSD0004(A2 R1) ._ Bs1WI AflIIpSD0050,pSD0051,pSD0052 (A1-R1)pSD0045, pSD0046, pSD0049, pSD0050, BsiWI--AflIILSD0037 pSD0032 )pSD0051, pSD0052 (A1-R1)pSD0045, pSD0046, pSD0049,LSD0038 pSD0039 (a3) BamHI__ClaIpSD0050,pSD0051,pSD0052 )LSD0039 pSD0039 (a3) pSD0032, pSD0001, pSD0003 (AZ-R1) BamHI--C1aIpSD0045, pSD0046, pSD0049,LSD0040 pSD0040,pSD0010,pSD0041 (A3 R1) ___ ClaI XbaIpSD0050,pSD0051,pSD0052 (A1-R1)LSD0041 pSD0040, 0, pSD0041 (A3-R1) pSD0032, pSD0001, pSD0003 (AZ-R1) C1aI--XbaIpSD0062, pSD0063, pSD0043, pSD0044 pSD0045, pSD0046, pSD0049,LSD0042 CM Xbal__(A3-R2) pSD0050,pSD0051,pSD0052(Al-R1)3 ?” pSD0063’ pSD0043’ pSD0044 pSD0032, pSD0001, pSD0003 (A2-R1) C1aI--XbaILSD0044 F213???” pSD0063’ pSD0043’ pSD0044 pSD0040, 0, pSD0041 (A3-R1) PflMI+XbaILSD0045 pSD0039 (a3) pSD0040, pSD0010, pSD0041 (A3-R1) -C1aIpSD0062, 3, pSD0043,LSD0046 pSD0039 (a3) BamHI__ClaIpSD0044(A3_R2)LSD0047 pSD0046(A1-R1) pSD0001,pSD0003 (AZ-R1) BsiWI—-AflIILSD0048 pSD0045,pSD0051(A1-R1) pSD0003 (AZ-R1) BsiWI--AflII6 PCR product 300603 Domam andC BamHI+PflMItermim)pNL0007 PCR product ”13990300603 Domam andC C1aI--PflMItermim)pNL0008 PCR product ”139903009 (B Domam andC C1aI--PflMItermim)pNL0009 PCR product pSD0039 (a3 Domain) BamHI+AscIpNL0010 LSD0003.006 (B Domain and C termini) pNL0009 (a3 Domain) XbaI+AgeI Example 22: Construction of BDD FVIII sion vectors with 3-5 XTEN insertions atsites 18/26 403 745/1656 1720 1900 or 2332 FVHI-fusion constructs With XTEN insertions at sites 18/26, 403, 745/1656, 1720, 1900 or2332 were chosen to ine and te constructs with 3, 4, 5 or 6 XTEN ions.
Construction of BDD FVIII expression vectors with 3-5 XTEN insertions at sites 26, 403,1656, 1720, or 1900 The chosen constructs with single XTEN at the desired sites were: O, pSDOOOl,pSDOO39, pSDOOlO, and pSDOO62. Constructs with double XTENs at the desired sites includedLSD0005.002, LSDOO38.001, LSDOO40.002, LSDOO42.013, LSDOO39.010, LSDOO41.008,LSDOO43.008, LSDOO45.002, LSDOO46.002, and LSDOO44.002. ng blocks and restrictionenzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components weredigested with unique restriction enzymes. DNA of inserts and vectors were separated with 1% agarosegel and purified by Qiagen gel extraction kit. The insert and vector were ligated, and then transformedinto DH50t E. coli competent host cells. Four colonies for each construct were analyzed by RCA andDNA sequencing. Clones with desired XTEN insertions were minipreped. Restriction digestions werethen used to further confirm the integrity ofXTEN in each region. The amino acid and the encodingDNA sequences for the resulting CFXTEN fusion ns are listed in Table 21. The resulting uctswere numbered 7 to pSDOO92. uction of BDD FVIII expression vectors with 4-6 XTEN insertions at sites 18, 403,1656, 1720, 1900 or 2332 Constructs pSDOO77 to pSDOO92 served as building blocks to generate 4- to 6-XTENconstructs with insertions at 18, 403, 1656, 1720, 1900 and 2332. Building block constructs andction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosencomponents were digested with unique restriction enzymes. DNA of inserts and vectors were separatedwith 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were ligated, andthen ormed into DH50t E. coli competent host cells. Eight colonies for each construct were analyzedby colony PCR and DNA sequencing. Clones with desired XTEN ions were minipreped.
Restriction digestions were then used to further m the integrity ofXTEN in each region. The aminoacid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21.
The resulting constructs were numbered pBCO247 to 7, pNLOO22, 23, 24, 25, and 30 Construction of BDD FVIII expression vectors with 4-6 XTEN insertions at sites 18, 403,745, 1720, 1900 or 2332 ucts pBCO247 to pBCO252, pBCO255, pNLOO22 to pNLOO25 served as building blocksto generate 4- to 6-XTEN constructs with insertions at 18, 403, 745, 1720, 1900 and 2332. Buildingblock constructs and ction enzymes for cloning the constructs were chosen, as listed in Table 19below. Chosen components were digested with unique restriction enzymes. DNA of inserts and vectorswere separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector wereligated, and then ormed into DH50t E. coli competent host cells. Eight colonies for each constructwere analyzed by colony PCR and DNA sequencing. Clones with desired XTEN insertions wereminipreped. Restriction digestions were then used to further confirm the ity ofXTEN in eachregion. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins arelisted in Table 21. The resulting constructs were numbered pBCO25 8 to pBCO268.
Table 19: Cloning design for FVIII libraries with 3-5 XTEN insertions at sites 26, 403, 1656, 1720,or 1900Cfiljgza Insert components (XTEN ) Vi§¥E§T§22:31ts gignpSDOO77 pSDOOSO (Al-R1) LSDOO39.010 (AZ-R1, a3) BsiWI+AflII8 pSDOOlO (A3-R1) LSD0005.002 (Al-R1, A2-R1) Xba1pSDOO79 pSDOO62 (A3-R2) LSD0005.002 (Al-R1, A2-R1) ClaI--Xba1pSDOO8O pSDOOSO (Al-R1) LSDOO45.002 (a3, A3-R1) BsiWI—-Afl11pSDOO81 pSDOOSO (Al-R1) LSDOO46.002 (a3, A3-R2) BsiWI--AflIIpSDOO82 O (Al-R1) LSDOO44.002 (A3-R1, A3-R2) BsiWI--AflIIpSDOO83 pSDOOlO (A3-R1) LSDOO39.010 (AZ-R1, a3) ClaI--Xba1pSDOO84 pSDOO62 ) LSDOO39.010 (AZ-R1, a3) ClaI--Xba1pSDOO85 pSDOO62 (A3-R2) LSDOO41.008 (AZ-R1, A3-R1) PflMI--Xba1pSDOO86 2 (A3-R2) LSDOO45.002 (a3, A3-R1) PflMI--Xba1pSDOO87 LSDOO39.010 (AZ-R1, a3) LSDOO40.002 (Al-R1, A3-R1) NheI--Cla1pSDOO88 LSDOO39.010 (AZ-R1, a3) LSDOO42.013 (Al-R1, A3-R2) NheI--C1a1pSDOO89 LSDOO44.002 (A3-R1, A3-R2) LSD0005.002 (Al-R1, A2-R1) ClaI--Xba1pSDOO9O LSDOO44.002 , A3-R2) LSDOO38.001 (Al-R1, a3) ClaI--Xba11 LSDOO44.002 (A3-R1, A3-R2) LSDOO39.010 (AZ-R1, a3) Xba1pSDOO92 LSDOO44.002 (A3-R1, A3-R2) 7 (Al-R1, A2-R1, a3) Xba1pBC0247 pSDOO77 LSD0050.003 \heI--BstBIpBC0248 pSDOO78 LSD0050.003 \heI--BstBIpBC0249 pSDOO79 LSD0050.003 \heI--BstBIpBC0250 pSDOO8O 0.003 \heI--BstBI1 pSDOO82 0.003 \heI--BstBIpBC0252 pSDOO8O LSD0050.003 \heI--BstBIpBC0253 pSDOO87 LSD0050.003 \heI--BstBI4 8 LSD0050.003 \heI--BstBIpBC0255 pSDOO89 LSD0050.003 \heI--BstBIpBC0256 pSDOO9O LSD0050.003 \heI--BstBIpBC0257 pSDOO92 LSD0050.003 \heI--BstBIpNL0022 LSDOOO3.009 pSDOO83 XbaI--AgelpNL0023 LSDOOO3.009 pSDOO84 XbaI--AgelpNL0024 LSDOOO3.009 pSDOO85 XbaI--AgelpNL0025 LSDOOO3.009 pSDOO86 XbaI--AgelpNLOO3O LSDOOO3.009 pSDOO91 XbaI--AgelpBC0258 LSDOOO3.006 pBC0247 BamHI--Cla1pBC0259 LSDOOO3.006 pBC0248 BamHI--Cla1nggza Insert components (XTEN region) veg§¥Ec§?§:gfinlts sttrigignpBC026O LSDOOO3.006 pBC0249 BamHI--Cla1 Example 23: Construction of CFXTEN expression vectors with three or four XTENs: thefirst XTEN in the B domain, the second XTEN at the C-terminus, and the third or fourth XTENinsertion within the A1 or A2 or A3 domains ies of CFXTEN fusion proteins were constructed with three XTEN insertions bycombining coagulation-active clones with XTEN insertions in the Al, A2, or A3 domains and cloneswith XTEN inserted within the B domain and at the inus. Additional libraries were constructedwith a fourth XTEN added in the Al, A2, or A3 domains to select members of the 3 XTEN libraries. Thedesign of the cloning scheme is summarized in the table below. DNA was prepared for the inserts andvectors by restriction enzyme ion and agarose gel ation. After ligating the inserts with thecorresponding vectors, the ligated DNA mixture was used to transform DH50t competent E. coli hostcells. ormants were screened by RCA and sequencing to cover approximately 3-4 times thepotential library size. Unique clones were identified and mini-prepped. Three distinct restrictiondigestions were then used to further confirm the integrity of each XTEN. The amino acid and theencoding DNA ces for the resulting CFXTEN fusion proteins are listed in Table 21.
Table 20: Clonin desi n for FVIII libraries with 3 XTEN insertions at sites B domain C-terminiand A1/A2/A3 domainLibrary Insert components (XTEN Vector 0011119011911“ RestrictionID region) (XTEN region) enzymesLSD0003.006 (B domain and C- pSD0045, pSD0046, pSD0049, pSD0050,9 BamHI Ag“__1, pSD0052 (Al-R1)LSD0003.009 (B domain and C- pSD0045, pSD0046, pSD0049, pSD0050,O BamHI Ag“__pSD0051, pSD0052 (Al-R1)LSD0051 igfig3‘006 (B domam and C' pSD0032, pSD0001, 3 (AZ-R1) BamHI--AgelWO 22617 pSD0062, pSD0063, pSD0043,LSD0056 LSD0003.009 (B domam and C-termlm). . . C1aI+XbaIpSD0044 (A3-R2)pBC0274 pSD0009 (A3_R1) LSD0003.006 (B domain and C-teimini) C1aI--XbalpNL0004 (A3_R2) LSD0003.006 (B domain and C-teimini) C1aI--XbalpBC0276 pBC0280 (A1_R1) LSD0003.006 (B domain and C-teimini)pBC0277 pBC0281 (A2_R1) 3.006 (B domain and C-teimini)pBC0278 pBC0282 (A3_R1) LSD0003.006 (B domain and C-teimini) C1aI--Xbal.BC0279 .BC0283 A3 R2 LSD0003.006 B domain and C-teimini ClaI--XbalL09_01 pBC0284 (CT) 39135013555:: é89’ 290’ 291’ 292’ AgelL09_01 pSD0014 (CT) 39135013555:: :fgffgé89’ 290’ 291’ 292’ XbaI--AgelpBC0285, 286, 287, 288, 289, 290, 291, 292,L09_01 pSD0020 (CT) Xbal Agel__293 (B domain and A3_R2)Table 21: DNA and Amino Acid Se uences of FVIII-XTEN ConstructsConstruct Amino acid sequence disclosed as DNA sequence disclosed asName SEQ ID NO: SEQ ID NO:pBC01 14 595 596pBC0126 597 598pBC0127 599 600pBC0165 601 6023 603 6044 605 606pBC0166 607 608pBC0185 609 610pBC0167 61 1 612pBC0128 613 614pBC0168 615 616pBC0129 617 618pBC0169 619 620pBC0130 621 622pBC0131 623 624pBC0132 625 626pBC0170 627 628pBC0133 629 630pBC0171 631 6324 633 6342 635 636pBC0135 637 63 8pBC0149 639 640pBC0136 641 642pBC0137 643 644pBC0138 645 646pBC0139 647 648pBC0140 649 650pBC0173 651 652pBC0174 653 654pBC0175 655 656pBC0176 657 658pBC0177 659 660pBC0178 661 662pBC0141 663 664pBC0179 665 666pBC0180 667 668pBC0142 669 6703 671 672pBC0181 673 674pBC0182 675 676pBC0144 677 678pBC0145 679 680pBC0146 681 6821 683 684pSD0002 685 686pSD0003 687 688pSD0004 689 690pSD0005 691 692pSD0006 693 694pSD0007 695 696pSD0008 697 698pSD0009 699 700pSD0010 701 702pSD0011 703 704pSD0012 705 706pSD0013 707 708pSD0014 709 710pSD0017 711 712pSD0018 713 714pSD0019 715 7160 717 718pSD0015 719 720pSD0016 721 722pSD0021 723 724pSD0022 725 726pSD0023 727 728pSD0024 729 730pSD0025 731 7326 733 734pSD0027 735 736pSD0028 737 738pSD0029 739 7400 741 742pSD0031 743 744pSD0032 745 746pSD0033 747 748pSD0034 749 750pSD0035 751 752pSD0036 753 754pSD0037 755 756pSD0038 757 758pSD0039 759 760pSD0040 761 762pSD0041 763 764pSD0042 765 766pSD0043 767 768pSD0044 769 770pSD0062 771 772pSD0063 773 774pSD0045 775 776pSD0046 777 778 2012/046326pSD0047 779 7808 781 782pSD0049 783 784pSD0050 785 7861 787 788pSD0052 789 790pSD0053 791 792pSD0054 793 794pSD0055 795 796pSD0056 797 798pSD0057 799 800pSD0058 801 802pSD0059 803 804pSD0060 805 806pSD0061 807 808LSD0001.002 809 810LSD0001.005 811 812LSD0001.006 813 814LSD0001.011 815 816LSD0001.012 817 818LSD0001.013 819 820LSD0001.016 821 822LSD0001.021 823 824LSD0002.001 825 826LSD0002.002 827 828LSD0002.014 829 830LSD0003.004 831 8323.006 833 834LSD0003.009 835 836LSD0003.014 837 8384.010 839 840LSD0004.011 841 842LSD0004.014 843 844LSD0004.016 845 846LSD0004.022 847 848LSD0003.016 849 850LSD0005.002 851 852LSD0005.004 853 854LSD0005.005 855 856LSD0005.011 857 858LSD0005.018 859 860LSD0006.002 861 862LSD0006.005 863 864LSD0006.007 865 866LSD0006.011 867 868LSD0007.002 869 870LSD0007.004 871 872LSD0007.013 873 874LSD0008.001 875 876LSD0008.002 877 878LSD0008.006 879 880LSD0008.009 881 882LSD0008.017 883 884LSD0002.025 885 8862.013 887 8883.025 889 890LSD0004.025 891 892LSD0003.005 893 8947.008 895 896LSD0044.002 897 898LSD0044.005 899 900LSD0044.039 901 902LSD0044.022 903 904LSD0044.003 905 906LSD0044.001 907 908LSD003 8.001 909 910LSD003 8.003 911 912LSD003 8.008 913 914LSD003 8.012 915 916LSD003 8.013 917 918LSD003 8.015 919 920LSD0039.001 921 922LSD0039.003 923 924LSD0039.010 925 926LSD0045.001 927 928LSD0045.002 929 930LSD0042.014 931 9322.023 933 934LSD0042.006 935 936LSD0042.013 937 938LSD0042.001 939 940LSD0042.039 941 942LSD0042.047 943 944LSD0042.003 945 946LSD0042.004 947 948LSD0042.008 949 950LSD0042.038 951 952LSD0042.082 953 954LSD0042.040 955 956LSD0037.002 957 958LSD0037.009 959 960LSD0037.011 961 962LSD0047.002 963 964LSD0047.005 965 966LSD0048.007 967 968LSD0046.001 969 9706.002 971 972LSD0046.003 973 974LSD0040.011 975 976LSD0040.042 977 978LSD0040.002 979 980LSD0040.008 981 982LSD0040.021 983 984LSD0040.037 985 986LSD0040.046 987 988LSD0040.003 989 990LSD0040.006 991 992LSD0040.007 993 994VV()2013/122617 LSD0040.010 995 996LSD0040.039 997 998LSD0040.052 999 1000LSD0041.001 1001 10021.004 1003 1004LSD0041.006 1005 1006LSD0041.008 1007 1008LSD0041.010 1009 1010LSD0041.014 1011 1012LSD0041.016 1013 1014LSD0041.035 1015 1016LSD0043.001 1017 1018LSD0043.002 1019 1020LSD0043.005 1021 1022LSD0043.006 1023 1024LSD0043.007 1025 1026LSD0043.008 1027 1028LSD0043.015 1029 1030LSD0043.029 1031 1032LSD0043.043 1033 1034pSD0077 1035 1036pSD0078 1037 1038pSD0079 1039 1040pSD0080 1041 1042pSD0081 1043 1044pSD0082 1045 1046pSD0083 1047 1048pSD0084 1049 1050pSD0085 1051 1052pSD0086 1053 1054pSD0087 1055 1056pSD0088 1057 1058pSD0089 1059 1060pSD0090 1061 1062pSD0091 1063 10642 1065 10669.002 1067 1068LSD0049.008 1069 1070LSD0049.011 1071 1072LSD0049.012 1073 1074LSD0049.020 1075 10769.021 1077 1078LSD0050.002 1079 1080LSD0050.003 1081 1082LSD0050.007 1083 1084LSD0050.010 1085 10860.012 1087 1088LSD0050.014 1089 1090LSD0051.002 1091 1092LSD0051.003 1093 1094LSD0052.001 1095 1096LSD0052.003 1097 1098LSD0053.021 1099 1100LSD0053.022 1101 1102VV()2013/122617 53.024 1103 1104LSD0054021 1105 1106LSD0054025 1107 1108LSD0054026 1109 1110LSI)0055.021 1111 1112LSD0055022 1113 1114LSD0055026 1115 1116LSD0056021 1117 1118LSD0056024 1119 11206025 1121 1122*0\10001 1123 1124*01’ L0002 1125 1126’U/333/ L0003 1127 1128L0004 1129 1130L0005 1131 1132L0006 1133 1134"0’5 L0007 1135 113633/L0008 1137 1138L0009 1139 1140mm 1141 1142chmz44 1143 1144chmz45 1145 1146chmz46 1147 1148chmz47 1149 1150pBC0248 1151 1152chmz49 1153 1154pBC0250 1155 1156pBC0251 1157 1158chmzsz 1159 1160pBC0253 1161 1162pBCD254 1163 1164pBC0255 1165 1166pBC0256 1167 1168pBC0257 1169 1170ch0259 1171 1172pBC0260 1173 1174chmzéz 1175 1176pBC0263 1177 1178pBCD264 1179 1180pBC0266 1181 1182pBC0267 1183 1184pBC0268 1185 1186pN10016 1187 1188pN10017 1189 1190pN10018 1191 1192leoozz 1193 11943 1195 1196pN10024 1197 1198leoozs 1199 1200leooso 1201 1202LSD0057001 1203 1204LSD0057004 1205 1206LSD0057005 1207 1208LSD0057010 1209 1210VV()2013/122617 LSD0058003 1211 1212LSD0058005 1213 1214LSD0058006 1215 1216LSD0059.002 1217 1218LSD0059.003 1219 1220LSD0059.005 1221 1222LSD0059.006 1223 1224LSD0060001 1225 1226LSD0060003 1227 1228LSD0060004 1229 1230LSD006L002 1231 1232LSD006L007 1233 1234LSD006L008 1235 1236LSD006L012 1237 1238LSD0062001 1239 1240LSD0062002 1241 1242LSD0062006 1243 12442007 1245 1246LSD0063001 1247 1248LSD0063003 1249 1250LSD0063011 1251 1252LSD0064017 1253 1254LSD0064018 1255 1256LSD0064020 1257 1258LSD0064021 1259 1260LSD0065001 1261 1262LSD0065007 1263 1264LSD0065014 1265 1266LSD0066001 1267 1268LSD0066002 1269 1270LSD0066009 1271 1272LSD0066011 1273 1274LSD0067004 1275 1276LSD0067005 1277 1278LSD0067006 1279 1280LSD0067008 1281 1282LSD0068001 1283 1284LSD0068002 1285 1286LSD0068005 1287 1288LSD0068010 1289 1290LSD0069004 1291 1292LSD0069008 1293 1294LSD0070.003 1295 1296LSD0070.004 1297 1298LSD0070.005 1299 1300LSD007L001 1301 1302L002 1303 1304LSD007L008 1305 1306LSD0072.001 1307 13082.002 1309 1310LSD0072.003 1311 1312LSI)0073.002 1313 1314LSI)0073.004 1315 1316LSD0073.006 1317 1318VV()2013/122617 LSD0074.007 1319 1320LSD0074.010 1321 1322LSD0074.01 1 1323 1324LSD0075.003 1325 1326LSD0075.004 1327 1328LSD0075.007 1329 1330LSD0076.002 1331 1332LSD0076.003 1333 1334pSD0093 1335 1336pSD0094 1337 1338pSD0095 1339 1340pSD0096 1341 1342pSD0097 1343 1344pSD0098 1345 1346pSD0099 1347 1348pSD0100 1349 1350pSD0101 1351 1352pSD0102 1353 13543 1355 1356pSD0104 1357 1358pcs0001 1359 1360pcs0002 1361 1362pcs0003 1363 1364pcs0004 1365 1366pcs0005 1367 13686 1369 1370pBC0269 1371 1372pBC0270 1373 1374pBC0271 1375 1376pBC0272 1377 1378pBC0273 1379 1380pBC0274 1381 1382pBC0275 1383 1384pBC0276 1385 1386pBC0277 1387 13888 1389 1390pBC0279 1391 1392pBC0280 1393 13941 1395 1396pBC0282 1397 1398pBC0283 1399 1400pBC0284 1401 1402pBC0285 1403 1404pBC0286 1405 1406pBC0287 1407 1408pBC0288 1409 1410pBC0289 1411 1412pBC0290 1413 1414pBC0291 1415 1416pBC0292 1417 1418pBC0293 1419 1420pBC0294 1421 1422pBC0295 1423 1424pBC0296 1425 1426pBC0297 1427 1428pBC0298 1429 1430pBC0299 1431 1432pBC0300 1433 1434pBC0301 1435 1436pBC0302 1437 1438pBC0303 1439 1440pBC0304 1441 1442pBC0305 1443 1444pBC0306 1445 1446pBC0307 1447 14488 1449 1450pBC0309 1451 1452pBC0310 1453 1454pBC0311 1455 1456pBC0312 1457 1458pBC0313 1459 1460pBC0314 1461 1462pBC0315 1463 1464pBC0316 1465 1466pBC0317 1467 1468pBC0318 1469 1470pBC0319 1471 1472pBC0320 1473 1474pBC0321 1475 1476PBC0322 1477 1478PBC0323 1479 1480pNL0040 1481 1482pNL0041 1483 1484pNL0042 1485 1486pNL0043 1487 1488 Example 24: Transfection of Mammalian Cells, Expression of FVIII-XTEN andAssessment of FVIII Activity ian cells, including but not limited to CH0, BHK, COS, and HEK293, are suitable fortransformation with the vectors of the Examples, above, in order to express and recover FVIII-XTENfusion protein. The following are details for methods used to express BDD FVlll and FVIll-XTENfusion protein constructs pBCOl l4, 5, pBCOl36, pBCOl37, pBCOl45, pBCOl46, and pBCOl49by transient transfection, which includes electroporation and chemical (PEI) transfection methods.
Adherent HEK293 cells purchased from ATCC were revived in medium of vendor’sendation and passaged for a few generations before multiple vials were frozen in the mediumwith 5% DMSO. One vial was d and ed one more time before transfection. The HEK293cells were plated 1-2 days before ection at a density of approximately 7X 105 per ml in one T175per ection, using 35 ml medium. On the day of transfection the cells were nized, detached andcounted, then rinsed in the medium until an even cell suspension was achieved. The cells were countedand an riate volume of cells (based on cell count above) were transferred to 50mL centrifuge tube,WO 22617 such that there were approximately 4 X 10° cells per transfection. Cells were centrifuged for 5min at 500RCF, the supernatant discarded, and the cells resuspended in 10ml of D-PBS.
Electroporation: For electroporation, an appropriate volume of resuspension buffer was addedusing a micropipette (supplied in the NeonTM Transfection System 100 uL Kit), such that 110 pl of bufferwas available per transfection. te volumes of 110 pl of cell suspension were added to eachEppendorf tube containing 11 ul of plasmid DNA for each of the individual FVIII-XTEN constructs for atotal of 6 ug (volume of DNA may be less, qs to 11 ul with sterile H20). A NeonTM ection Devicewas used for transfection. The program was set to electroporate at 1100v for a pulse width of 20ms, for atotal of two pulses. A NeonTM Tube (supplied in the NeonTM Transfection System 100 uL Kit) wasplaced into NeonTM Pipette Station. A volume of 3 mL of Electrolytic Buffer E2 ied in the NeonTMTransfection System 100 uL Kit) was added to the NeonTM Tube. NeonTM Pipettes and 100 pl NeonTMTips were used to electroporate 100 pl of cell-plasmid DNA mixture using the NeonTM Pipette Station.
The electroporation was executed and when complete, the NeonTM e was removed from the Stationand the pipette with the transfected cells was used to transfer the cells, with a circular motion, into a 100mm x 20mm petri plate containing 10 ml of Opti-MEM I Reduced-Serum Medium (1X, Invitrogen), suchthat transfected cells were evenly distributed on plate. The cells for each ection were incubated at37°C for expression. On day 3 post-transfection, a 10% volume of salt solution of 10mM Hepes, 5mMCaClg, and 4M NaCl was added to each cell e and gently mixed for 30 minutes. Each cell culturewas transferred to a 50 ml conical centrifuge tube and was centrifuged at 3000 rpm for 10 minutes at 4°C.
The supematants for each culture were placed into a new 50 ml conical tube and then split into tsof 5x1 ml in Eppendorf and 2x15ml conical tubes for assay or were flash frozen before g forexpression of FVIII-XTEN in ELISA and performance in an FVIII activity assay, as described herein.
Chemical transfection: Chemical transfection can be accomplished using standard methodsknown in the art. In the present e, PEI is utilized, as described.
Suspension 293 Cells are seeded the day before transfection at 7 x 105 cells/mL in sufficientFreestyle 293 (Invitrogen) medium to provide at least 30 ml working volume, and incubated at 37°C. Onthe day of transfection, an aliquot of 1.5 ml of the transfection medium is held at room temperature, towhich 90 uL of lmg/ml PEI is added and vortexed briefly. A volume of 30 ul of DNA encoding theFVIII-XTEN_AE288 construct (concentration of lmg/ml) is added to the PEI solution, which is vortexedfor 30 sec. The mixture is held at room temperature for 5-15 min. The I mixture is added to theHEK293 cells and the suspension is incubated at 37°C using pre-established shake flask ions.
About four hours after the addition of the DNA/PEI mix, a 1x volume of expansion media is added andthe cells incubated at 37°C for 5 days. On the day of harvest, a 10% volume of salt solution of 10mMHepes, 5mM CaClg, and 4M NaCl is added to the cell culture and gently mixed for 30 minutes. The cellculture is erred to a 50 ml conical centrifuge tube and is centrifuged at 4000 rpm for 10 minutes at4°C. The supernatant is placed into a new 50 ml conical tube and then split into aliquots of 5x1 ml inEppendorf and 2x15ml conical tubes for assay or are flash frozen before testing for expression of FVIII-XTEN in ELISA and/or performance in an FVIII activity assay, as bed herein.
] Generation of stable pools and cell lines that produce FVIII-XTEN Stable pools are generated by culturing transfected cells for 3-5 weeks in medium containingselection antibiotics such as puromycin, with medium change every 2-3 days. Stable cells can be usedfor either production or generation of stable clones. For stable cell line selection during primaryscreening, cells from stable pools either from on—going passaging or revived from frozen vials are seededin 96-well plates at a target density of 0.5 cell/well. About 1 week after seeding spent medium fromwells with single cell cluster as observed under microscope are tested for expression of FVIII by tyassay or antigen measurement.
For additional rounds of screening, normalized numbers of cells are seeded in multi-well plates.
Spent medium is harvested and tested for FVIII tration by ELISA and FVIII activity assay. Cellswould also be ted from the plates and counted using Vi-Cell. Clones are ranked by (l) FVIII titersaccording to ELISA and activity; (2) ratios of ELISA titer/cell count and activity titer/cell count; and (3)integrity and homogeneity of products produced by the clones as measured by n blots. A numberof clones for each of the constructs are ed from the primary screening for additional rounds ofscreening.
For the second round of screening, cells in 96-well plates for the top clones ed fromprimary screening are first expanded in T25 flasks and then seeded in duplicate 24-well plates. Spentmedium is ted from the plates for FVIII activity and antigen quantification and cells harvested andcounted by Vi-Cell. Clones are ranked and then selected according to titers by ELISA and activity assay,ELISA titer/cell and activity titer/cell count ratios. Frozen vials are prepared for at least 5-10 clones andagain these clones were screened and ranked according to titers by ELISA and activity, and ratios ofELISA titer/cell count and activity titer/cell count, and product integrity and homogeneity by Westernblot, and 2-3 clones are selected for productivity evaluation in shake flasks. Final clones are selectedbased on specific productivity and product quality.
Production of FVIII-XTEN secreted in cell culture medium by sion 293 stable HEK293 stable cell clones selected by the ing methods are seeded in shake flasks at 1-2X 105 cells/ml in expression medium. Cell count, cell viability, FVIII activity and n expressiontiters are monitored daily. On the day when FVIII activity and antigen titers and product quality areoptimal, the culture is ted by either centrifugation/sterile filtration or depth filtration/sterileion. The filtrate is either used immediately for tial flow filtration (TFF) processing andpurification or stored in -80°C freezer for TFF processing and purification later.
Example 25: Purification and Characterization of CFXTEN Constructs Exemplary methods for the purification and characterization of CFXTEN constructs with oneor more XTEN follow.
Purification of FVII-XTEN AE864 by FVIII affinity chromatography CFXTEN containing supernatant is filtered using a Cuno ZetaPlus Biocap filter and a CunoBioAssure capsule and subsequently concentrated by tial flow filtration using a ore Pellicon2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration cartridge thesample is ered into 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and0.02% Tween 80 at pH 7.0. lect resin (GE 1701) selectively binds FVIII or B domaindeleted FVIII using a 13kDa recombinant protein ligand coupled to a chromatography resin. The resin isequilibrated with 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02%Tween 80 at pH 7.0 and the supernatant loaded. The column is washed with 20 mM histidine, 20 mMcalcium de, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0, then is washed with 20 mMhistidine, 20 mM calcium chloride, 1.0 M sodium chloride, and 0.02% Tween 80 at pH 7.0, and elutedwith 20 mM ine, 20 mM calcium chloride, 1.5 M sodium chloride, and 0.02% Tween 80 dissolvedin 50% ethylene glycol at pH 7.0.
Concentration and Buffer Exchange by Tangential Flow Filtration and Diafiltration Supernatant batches totaling at least 10 L in , from stable CHO cells lines expressingCFXTEN are filtered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssure capsule. They aresubsequently concentrated approximately 20-fold by tangential flow ion using a Millipore Pellicon2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration cartridge the sampleis diafiltered with 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02%Tween 80 at pH 7.0 10 mM tris pH 7.5, 1 mM EDTA with 5 volumes worth of buffer exchange.
Samples are divided into 50 ml aliquots and frozen at -80°C.
Purification of CFXTEN by Anion Exchange Chromatography Using an Akta FPLC system the sample is purified using a SuperQ-650M column. The columnis equilibrated into buffer A (0.02 mol/L imidazole, 0.02 mol/L glycine ethylester hydrochloride, 0.1 5mol/L, NaC1, 2.5% glycerol, pH 6.9) and the sample . The sample is eluted using buffer B (5mmol/L histidine HCl (His/HCI), 1.15 mol/L NaCl, pH 7.0). The 215 nm chromatogram is used tomonitor the elution . The eluted fractions are assayed for FVIH by ELISA, SDS-PAGE or activityassay. Peak fractions are pooled and stored or subjected to thrombin activation immediately (O’Brien etal., Blood (1990) 75:1664-1672). Fractions are assayed for FVIH activity using an aPTT based factorassay. A Bradford assay is performed to ine the total amount of protein in the load and elutionfractions.
] Purification of CFXTEN by Hydrophobic ction Chromatography CFXTEN samples in Buffer A (50 mmol/l histidine, 1 mmol/l CaCl 2, 1 M NaCl, and 0.2 g/lTween 80®, pH 7.0) are loaded onto a toyopearl ether 650M resin equilibrated in Buffer A. The columnis washed with 10 column volumes of Buffer A to remove DNA, incorrectly folded forms and FVIH, andother contaminant proteins. The CFXTEN is eluted with Buffer B (25 mmol/l histidine, 0.5 mmol/l CaCl2 and 0.4 mol/l NaCl, pH 7.0) as a single step elution (US patent 6005082). Fractions are assayed forFVIH activity using an aPTT based factor assay. A Bradford assay is performed to determine the totalamount of n in the load and elution fractions.
Removal of Aggregated protein from monomeric CFXTEN with Anion ExchangeChromatography Using an Akta FPLC system the sample is purified using a macrocap Q column. The column isbrated into buffer A (20 mM MES, lmM CaCl2, pH 7.0) and the sample is loaded. The sample iseluted using a linear gradient of 30% to 80% buffer B (20 mM MES, lmM CaC12, pH 7.0 + 500 mMNaCl) over 20 column volumes. The 215 nm chromatogram is used to monitor the elution profile. Thefractions corresponding to the early portion of the elution contain primarily monomeric protein, while thelate portion of the elution contains primarily the aggregated species. Fractions from the macrocapQcolumn is analyzed via size exclusion chromatography with 60 cm BioSep G4000 column to determinewhich to pool to create an aggregate free sample.
Activation of FVIII by Thrombin Purified FVIII in 5 mmol/L histidine HCl (His/HCI), 1.15 mol/L NaCl, pH 7.0 is treated withthrombin at a 1:4 ratio of units of human thrombin to units FVIII, and the sample is incubated at 37°C forup to 2 hours. To monitor the tion s, aliquots of this sample are then withdrawn, and acetoneprecipitated by the addition of 4.5 vol ice-cold acetone. The sample is incubated on ice for 10 minutes,and the precipitate is ted by centrifugation at 13,000 g in a microfuge for 3 minutes. The acetone isremoved, and the precipitate is resuspended in 30 ML SDS-PAGE reducing sample buffer and boiled for2 minutes. Samples are then assayed by SDS-PAGE or western blot. The conversion of FVIII t0 FVIIIais ed by looking for the conversion of the heavy chain into 40 and 50 kDa fragments and theconversion of the light chain into a 70 kDa nt (O’Brien et al., Blood (1990) 4-1672).
SEC Analysis of CFXTEN] FVII-XTEN purified by affinity and anion exchange chromatography is analyzed by sizeexclusion chromatography with 60 cm BioSep G4000 column. A monodispersed population with ahydrodynamic radius of ~10 nm/ apparent MW 0f~1.7 MDa (XTEN—288 fusion) or ~12 nm/ annt MW of 5.3 MDa (XTEN—864 fusion) is indicative of an ation-free sample. CFXTEN ised to have an apparent molecular weight factor up to or about 8 (for an XTEN—288 fusion withFVIII) or up to or about ~15 (for an XTEN—864 fusion with FVIII).
ELISA based Concentration Determination of CFXTEN] The quantitative determination of factor VIII / CFXTEN antigen concentrations using thedouble antibody enzyme linked immuno-sorbent assay (ELISA) is med using proven antibodygs (VisuLizeTM FVIII Antigen kit, Affinity Biologicals, Ontario Canada). Strip wells are pre-coated with sheep polyclonal antibody to human FVIII. Plasma samples are diluted and applied to thewells. The FVIII antigen that is t binds to the coated antibody. After washing away unboundmaterial, peroxidase-labeled sheep detecting antibody is applied and allowed to bind to the edFVIII. The wells are again washed and a solution of TMB (the peroxidase substratetetramethylbenzidine) is applied and d to react for a fixed period of time. A blue color developswhich changes to yellow upon quenching the reaction with acid. The color formed is measuredspectrophotometrically in a microplate reader at 450 nm. The absorbance at 450 nm is directlyproportional to the quantity of FVIII antigen captured onto the well. The assay is calibrated using eitherthe calibrator plasma provided in the kit or by substituting a CFXTEN standard in an appropriate matrix.
Assessment of CFXTEN Activity via a FXa Coupled Chromogenic ate Assay Using the ChromogeniX Coamatic Factor VIII (ChromogeniX, cat# 82258563) the activity ofFVIII or CFXTEN comprising FVIII is assessed as follows. In the presence of calcium ions andphospholipids, factor X is activated to factor Xa by factor IXa. This activation is greatly stimulated byfactor VIII which acts as a cofactor in this reaction. By using optimal s of Ca2+, phospholipid andfactor IXa, and an excess of factor X, the rate of activation of factor X is linearly related to the amount offactor VIII. Factor Xa hydrolyses the chromogenic substrate S-2765 thus liberating the chromophoricgroup, pNA. The color is then read spectrophotometrically at 405 nm. The generated factor Xa and thusthe ity of color is proportional to the factor VIII actiVity in the sample. Hydrolysis of S-2765 bythrombin formed is prevented by the addition of the synthetic thrombin inhibitor I-2581 together with thesubstrate. The actiVity of an unknown sample is determined by comparing final A405 of that sample tothose from a standard curve constructed from known FVIII amounts. By also determining the amount ofFVIII antigen present in the samples (Via A280 or ELISA), a specific actiVity of a sample is ine totand the relative potency of a particular preparation of FVIII. This enables the relative ncyof ent isolation strategies or construct designs for CFXTEN fusions to be assessed for activity andranked.] aPTT Based Assays for CFXTEN Activity Determination CFXTEN acts to replace FVIII in the sic or contact activated coagulation pathway. TheactiVity of this coagulation pathway is assessed using an activated partial thromboplastin time assay. FVIII activity specifically is ed as follows: a standard curve is prepared by dilutingnormal control plasma (Pacific Hemostasis cat# 100595) ld with FVIII deficient plasma (cat#100800) and then conducting 6, 4-fold serial dilutions again with factor VIII deficient plasma. Thiscreates a standard curve with points at 500, 130, 31, 7.8, 2.0, 0.5 and 0.1 IU/ml of activity, where oneunit of actiVity is defined as the amount of FVIIIC actiVity in 1 ml of normal human plasma. A FVIII-deficient plasma also is included to determine the background level of actiVity in the null plasma. Thesample is prepared by adding CFXTEN to FVIII deficient plasma at a ratio of 1:10 by volume. Thesamples is tested using an aPTT assay as follows. The samples are incubated at 37C in a moleculardevices plate reader spectrophotometer for 2 minutes at which point an equal volume of aPTT reagent(Pacific Hemostasis cat# 100402) is added and an additional 3 minute 37C incubation performed. Afterthe incubation the assay is activated by adding one volume of calcium chloride (Pacific asis cat#100304). The turbidity is monitored at 450 nm for 5 minutes to create reaction s. The aPTT time,or time to onset of ng activity, is defined as the first time where OD405 nm sed by 0.06 overbaseline. A log — linear standard curve is created with the log of ty relating linearly to the aPTTtime. From this the actiVity of the sample in the plate well is determined and then the actiVity in thesample is determined by multiplying by 11 to account for the dilution into the FVIII deficient plasma.
By also determining the amount of FVIII antigen present in the samples (Via A280 or ELISA), a specificactivity of a sample can be determine to tand the relative potency of a particular preparation ofFVIII. This enables the ve efficiency of different isolation strategies or construct designs forCFXTEN fusions to be ranked.
Western Blot Analysis of FVIII / FVIII-XTEN expressed proteins Samples were run on a 8% homogeneous SDS gel and uently transferred to PVDFmembrane. The samples in lanes l-15 were: MW Standards, FVIII(42.5 ng), pBC0100B, pBC0114A,pBC0100, pBC0114, pBC0126, pBC0127 (8/5/11; #9), pBC0128, pBC0135, pBC0136, pBC0137,, pBC0149, and pBC0146, respectively. The membrane was initially blocked with 5% milk thenprobed with anti-FVIII monoclonal antibody, 2, specific to the A2 domain of the heavy chain(Ansong C, Miles SM, Fay PJ.J Thromb Haemost. 2006 Apr;4(4):842-7). Insertion ofXTEN288 in theB-domain was observed for 6 (lane 8, ) and pBC0137 (lane 9, ), whereasXTEN288 insertion at the C-terminus was observed for pBC0146 (lane 12, ). All of the assayedFVIII-XTEN proteins revealed the presence of single chain protein with molecular weight of at least 21kDa higher than that of pBC01 14 base uct or FVIII standard. In on, AE42 ion wasobserved for pBC0135 (lane 7, ) and pBC0149 (lane 11, ) with the single chain runningNS kDa higher than that of pBC01 14 base protein and heavy chain running at NS kDa higher than 90 kDaband of the base protein.
Assay of sed FVIII by ELISA To verify and quantitate the expression of FVIII-XTEN fusion proteins of the constructs by cellculture, an ELISA assay was established. e antibodies, either SAF8C-AP (Affinity Biologicals),or GMA—8002 (Green Mountain Antibodies), or GMAOll dies (Green Mountain Antibodies) forFVIII-LC ELISA) or by GMA016 antibodies were immobilized onto wells of an ELISA plate. The wellswere then incubated with blocking buffer (lx PBS/3% BSA) to prevent non-specific binding of otherproteins to the anti-FVIII antibody. FVIII standard dilutions (~50 ng-0.024 ng range), quality controls,and cell culture media samples were then incubated for 1.5 h in the wells to allow binding of thesed FVIII protein to the coated antibody. Wells were then washed extensively, and bound nis incubated with anti-FVIII detection antibody, SAF8C-Biotinylated (Affinity Biologicals). ThenstreptaVidin-HRP, which binds the biotin ated to the FVIII detection antibody, is added to the welland incubated for l h. Finally, OPD ate is added to the wells and its hydrolysis by HRP enzyme ismonitored with a plate reader at 490 nm wavelength. Concentrations of FVIII-containing samples werethen calculated by comparing the colorimetric response at each culture dilution to a standard curve. Theresults, in Table 22, below, show that FVIII-XTEN of the various constructs are expressed at 0.4 - l[Lg/ml in the cell culture media. The results obtained by ELISA and the actiVity data indicate that FVIII-XTEN fusion ns were very well expressed using the described transfection methods. Furthermore,under the experimental conditions, the results demonstrate that the specific actiVity values of the FVIII-XTEN proteins were similar or greater than that of pBC01 14 base construct (expressing BDD FVIII) andsupport that XTEN insertion into the C-terminus or B-domain of FVIII results in preservation of FVIIIprotein function.
Chromogenic Activity Assay for CFXTEN fusion n BDD FVIII and CFXTEN fusion n constructs pBC0114, 5, pBC0136, pBC0137,pBC0145, pBC0146, and pBC0149, in various configurations, including XTEN AE288 and AG288inserted at the C-terminus of the FVIII BDD ce and FVIII-XTEN fusion proteins with AE42 andAE288 inserted after residue 745 (or residue 743) and before residue 1640 (or residue 1638) of the B-domain (including constructs with the P1648 sing site d to alanine), were expressed intransiently transfected Freestyle 293 cells, as described above, and tested for procoagulant activity. Theprocoagulant activity of each of the FVIII-XTEN proteins present in cell e medium was assessedusing a Chromogenix Coamatic® Factor VIII assay, an assay in which the activation of factor X waslinearly related to the amount of factor VIII in the sample. The assay was performed according tomanufacturer’s instructions using the end-point method, which was measured ophotometrically atOD405 nm. A standard curve was created using purified FVIII protein at concentrations of 250, 200,150, 100, 75, 50, 37.5, 25, 12.5, 6.25, 3.125 and 1.56 mU/ml. Dilutions of factor VIII rd, qualitycontrols, and samples were prepared with assay buffer and PEI culture medium to account for the effectof the medium in the assay performance. Positive controls consist of purified factor VIII protein at 20,40, and 80 mU/ml concentrations and cell culture medium of pBC01 14 FVIII base construct, lacking theXTEN insertions. Negative controls consisted of assay buffer or PEI culture medium alone. The cellculture media of the FVIII-XTEN ucts were ed as described, above, and were tested inreplicates at 1:50, 1:150, and 1:450 dilutions and the actiVity of each was calculated in U/ml. Each FVIII-XTEN construct exhibited procoagulant actiVity that was at least comparable, and in some cases greaterthan that of the base construct ve l, and support that under the conditions of the experiments,the linkage of XTEN, including AE288 or AG288, at the C-terminus of FVIII or insertion of XTEN,including AE42 or AE288 within the B-domain resulted in retention or even enhancement of FVIIIprocoagulant actiVity=Table 22: Results of ELISA and Chromogenic FVIII activity assaysFVIII-Act1v1ty. . Concentration. Specrfic Actmty. . .
XTEN Description of Construct(IU/ml) (IU/mg)ConstructBDD FVIII base construct used forFVIII construct with XTE\I AG288pBC0146 -“ 12759inserted at the C-terminus of FVIIIFVIII construct with XTE\I AE288pBC0145 -“ 4844ed at the C-terminus of FVIIIFVIII construct with XTE\I AE42inserted between residue 745 and 1640pBC0149 4.9 55 81 ed between residue 745 and 1640and with Ar1648 to Ala mutationFVIII construct with XTE\I AE288inserted between residue 745 and 1640pBC0137 1.9 0.3 6013 inserted between residue 745 and 1640and with 8 to Ala mutation Coatest Assay for Cell e Sample Activity Assay containing CFXTEN fusion protein Using the Coatest assay, the activity of FVIII or CFXTEN comprising FVIH is assessed asfollows.
Assay Matrix: All wells in the same plate were adjusted to the same percentage of media tocontrol for matrix effects. The test s were diluted such that the OD405 reading would fall withinthe linear range of the standard. The range of concentrations for the FVIH standard was 100 mU/mL to0.78 mU/mL, prepared by four-fold serial dilutions of the FVlll standard in 1X Coatest buffer(DiaPharma) plus the pre-determined percentage of culture media.
The Coatest SP FVIII (DiaPharma) reagent package includes the 10X Coatest buffer stocksolution, factor lXa + factor X, phospholipid, nd substrate. The 1x Coatest solution was preparedby adding 9X volume of cold dngO to 1X volume of the stock. The cell culture media was then addedto the prepared 1X on at a pre-determined ratio to normalize the percentage of matrix in all testwells. Factor lXa + factor X, phospholipid, and substrate were reconstituted according tocturer’s recommendations.
Coatest Assay Procedure: Assay reagents were ed and kept on ice until . 25 [ll of the diluted test samplesand standards were added to a 96 well plate in duplicate. 50 pl of phospholipid/factor lXa/factor X wasadded to each well and mixed by gently tapping the side of the plate. Plates were incubated at 37°C for 5min on a 37°C plate heater. 25 pl of C3C12 was added to each well and mixed. The plates wereincubated at 37°C for 5 min on a plate heater. 50 ul of substrate was then added to each well, mixed, andthe plates incubated at 37°C for an onal 5-10 min until the top standard developed an OD405reading of about 1.5. 25 [ll of 20% acetic acid was added to each well with mixing to stop the reactionand wells were read at OD405 using a SpectraMAX® plus (Molecular Devices) spectrophotometer. Dataanalysis was performed using the x program (verion 5.2). The LLOQ varied per assay, but wasgenerally 0.0039 lU/ml.
: The data are presented in Tables 23-26. Table 23 presents results from CFXTENfusion proteins wih XTEN inserted in single sites chosed on the basis of criteria described herein,including Example 34. The pBC00114 FVIII positive control showed good expression and FVIII activity.
Of the 106 single-XTEN fusion proteins assayed, 68% retained measurable FVIH activity, with 30%exhibiting 3+ to 4+ activity in the coagulation assay. -one percent of the fusion proteins assayedhad results below the limits of quantitation (which may be due to poor expression, reflected in thecorresponding expression ELISA results). All four B-domain insertion ucts exhibited goodactivity, as did the inal linked constructs, indicating that these are likely favorable insertion sites The s of the single insertion site data guided the creation ofXTEN constructs with 2XTEN insertions, the results of which are presented in Table 24. Overall, the positivity rate was 67%,with 31% of fusion proteins exhibiting 3+ to 4+ activity in the coagulation assay. 2012/046326 The results of the foregoing data guided the creation of XTEN constructs With 3 XTENinsertions, the results of Which are presented in Table 25. Overall, 92% of the samples had measurableFVIII activity, With fully 79% exhibiting 3+ to 4+ activity in the coagulation assay.
A limited number of constructs With 4 XTEN inserted in the Al, A2 and A3 domains werecreated and assayed, With 4 of 5 ting FVIH actiVity (Table 26), ting that insertion of multipleXTEN does not compromise the ability of the resulting fusion proteins to retain FVIII actiVity.
Conclusions: Under the ions of the experiments, the results t that the criteria usedto select XTEN ion sites are valid, that insertion of one or more XTEN into the selected sites ofFVIII is more likely than not to result in retention of gulant actiVity of the resulting CFXTENmolecule, and that insertion of three XTEN appears to result in a greater proportion of fusion proteinsretaining high levels of FVIII procoagulant actiVity compared to single or double XTEN insertionconstructs.
Table 23: Results of Coagulation Activity Assays for CFXTEN comprising one XTEN11188652011 Domain Construct Activity 13%;???“pBC01 14 +——+ +——+3 A1 pBC0126 LLOQ* LLOQ3 A1 pBC0127 —— ——18 A1 pBC016522 A1 pBC0183 +——+26 A1 pBC018440 A1 pBC016660 A1 pBC0185 LLOQ LLOQ1 16 A1 pBC0167 LLOQ LLOQ130 A1 pBC0128 LLOQ LLOQ188 A1 pBC0168216 A1 pBCO 129230 A1 pBC0169 LLOQ LLOQ333 A1 pBC01303 75 A2 pBC013 1 LLOQ +++403 A2 pBC0132442 A2 pBC0170490 A2 pBC0133 ——518 A2 pBC0171 LLOQ --599 A2 pBC0134 ++713 A2 pBC0172 ——745 B pBC0135745 B 9745 B pBC0136 ++ ++745 B pBC01371720 A3 pBC013 81796 A3 pBC0139 ——1802 A3 pBCO 140 ——1827 A3 pBC0173 LLOQ LLOQ1861 A3 pBC0174 LLOQ LLOQ1896 A3 5 LLOQ LLOQ1900 A3 pBC0176 +——+ +——+1904 A3 pBC0177 —— --VV()2013/122617 Inserfion ExpressionDomain uct ActivitySite ELISA1937 A3 pBC0178 LLOQ LLOQ2019 A3 pBC0141 LLOQ +2068 C1 chnl79 ++ ++2111 C1 chnlso LLOQ LLOQ2120 C1 chnl42 LLOQ2171 C2 pBC01432188 C2 pBC01812227 C2 chnlsz2277 C2 chn1442332 CT chnl452332 CT chn146403 A2 pSD0001599 A2 pSD0002403 A2 pSD0003599 A2 pSD0004745 B pSD0005745 UUUUUU pSD0006745 pSD0007745 pSD00081720 A3 pSD00091720 A3 pSD00102171 C2 pSD00112171 C2 pSD00122332 CT‘ 32332 CT‘ 4745 0303 pSD0017745 pSD00182332 CT‘ pSD00192332 CT‘ pSD00202332 CT‘ pSD00152332 CT‘ pSD00160 1J4enn pSD002132 A1 pSD002265 A1 pSD002381 A1 pSD0024119 A1 pSD0025211 A1 pSD0026220 A1 pSD0027224 A1 pSD0028336 A1 pSD0029339 A1 pSD0030378 A2 pSD0031 LLOQ399 A2 2409 A2 3416 A2 pSD0034487 A2 pSD0035 LLOQ494 A2 pSD0036 LLOQ500 A2 pSD0037 LLOQ603 A2 pSD00381656 A3 pSD0039 +——+1656 A3 pN1009** ++++ 2012/046326Insertion ExpressionDomain Construct tySite ELISA1711 A3 pSD0040 ++ +1725 A3 pSD0041 LLOQ1749 A3 pSD0042 LLOQ LLOQ1905 A3 pSD00431910 A3 pSD0044 +1900 A3 21900 A3 pSD006318 A1 pSD004518 A1 pSD004622 A1 pSD0047 LLOQ LLOQ22 A1 pSD0048 LLOQ LLOQ26 A1 pSD004926 A1 pSD005040 A1 pSD005140 A1 pSD0052216 A1 pSD0053 LLOQ216 A1 pSD0054 LLOQ375 A2 pSD0055 LLOQ442 A2 pSD0056 LLOQ442 A2 pSD0057 LLOQ1796 A3 pSD0058 LLOQ1796 A3 pSD00591802 A3 pSD00601802 A3 1 LLOQ*LLOQ: below the limits of quantitation** pNL009 includesa deletion of 745-1656Table 24: Results of ation Activity Assays for CFXTEN comprising two XTENInsertion 1 Insertion 2Insertion InsertionDomain ADomain Construct ActivitySite Site745 Dd 2332 CT LSD0001.002 +++745 2332 CT 1.005 +++745 2332 CT LSD0001.006 +++745 2332 CT LSD0001.011 +++745 2332 CT LSD0001.012 +++745 2332 CT LSD0001.013 +++745 2332 CT LSD0001.016 +++745 LSD0001.021 2332 CT +++745 2332 CT LSD0002.001 +++745 wwwwwwwwwwwwwwwwww 2332 CT LSD0002.002 +++745 2332 CT LSD0002.014 +++745 2332 CT LSD0003.004 +++745 2332 CT LSD0003.006 +++745 2332 CT LSD0003.009 +++745 2332 CT LSD0003.014 +745 2332 CT LSD0004.010 +++745 2332 CT LSD0004.011 LLOQ745 2332 CT LSD0004.014 +++745 2332 CT LSD0004.016 +++Insertion 1 Insertion 211156111011 Domain Insnrnon Domain Construct Activity8116 Sam745 B 2332 CT LSD0004.022 +++745 B 2332 CT 3.016 +++0745 B 2332 CT pNLOO60745 B 2332 CT pNLOO70745 B 2332 CT pNLOO8 ++1656 213 2332 CT pNLOlO26 A1 403 A2 5.002 ++26 A1 403 A2 LSD0005.004 ++40 A1 403 A2 LSD0005.005 ++40 A1 403 A2 LSD0005.011 ++18 A1 403 A2 LSD0005.018 ++26 A1 599 A2 LSD0006.002 +40 A1 599 A2 6.005 ++40 A1 599 A2 LSD0006.007 ++40 A1 599 A2 LSD0006.011 +++40 A1 403 A2 LSD0007.002 +40 A1 403 A2 LSD0007.004 +26 A1 403 A2 LSD0007.013 ++26 A1 599 A2 LSD0008.001 ++40 A1 599 A2 LSD0008.002 ++26 A1 599 A2 LSD0008.006 +18 A1 599 A2 LSD0008.009 ++40 A1 599 A2 LSD0008.017 +745 B 2332 CT LSD0002.025 +++745 B 2332 CT 2.013 +++745 B 2332 CT LSD0003.025 +++745 B 2332 CT LSD0004.025 +++745 B 2332 CT LSD0003.005 ++26 A1 403 A2 LSD0007.008 ++1720 A3 1900 A3 LSD0044.002 LLOQ1725 A3 1900 A3 LSD0044.005 LLOQ1720 A3 1900 A3 LSD0044.039 LLOQ1711 A3 1905 A3 4.022 LLOQ1720 A3 1905 A3 LSD0044.003 LLOQ1725 A3 1905 A3 LSD0044.001 LLOQ1656 A3 26 A1 LSD003 8.001 ++1656 A3 18 A1 LSD003 8.003 ++1656 A3 18 A1 LSD0038.008 +++1656 A3 40 A1 LSD0038.012 ++1656 A3 40 A1 LSD0038.013 ++1656 A3 26 A1 LSD0038.015 ++1656 A3 399 A2 LSD0039.001 +1656 A3 403 A2 LSD0039.003 ++1656 A3 403 A2 LSD0039.010 ++1656 A3 1725 A3 LSD0045.001 +1656 A3 1720 A3 LSD0045.002 ++1900 A3 18 A1 LSD0042.014 +1900 A3 18 A1 LSD0042.023 +1900 A3 26 A1 LSD0042.006 +1900 A3 26 A1 LSD0042.013 ++1900 A3 40 A1 LSD0042.001 +1900 A3 40 A1 LSD0042.039 +Insertion 1 Insertion 2Insertion Domain Insertion Domain uct ActivitySite Site1900 A3 26 A1 LSD0042.047 +1905 A3 18 A1 LSD0042.003 +1905 A3 40 A1 LSD0042.004 LLOQ1905 A3 26 A1 LSD0042.008 LLOQ1905 A3 26 A1 LSD0042.038 LLOQ1905 A3 40 A1 LSD0042.082 LLOQ1910 A3 26 A1 LSD0042.040 LLOQ18 A1 399 A2 LSD0037.002 ++26 A1 399 A2 7.009 +40 A1 399 A2 LSD0037.011 ++18 A1 403 A2 LSD0047.002 ++18 A1 403 A2 LSD0047.005 +18 A1 403 A2 LSD0048.007 +1656 A3 1900 A3 LSD0046.001 ++1656 A3 1900 A3 LSD0046.002 +1656 A3 1905 A3 LSD0046.003 +1711 A3 40 A1 LSD0040.011 LLOQ1711 A3 26 A1 0.042 LLOQ1720 A3 26 A1 LSD0040.002 +1720 A3 40 A1 LSD0040.008 +1720 A3 18 A1 LSD0040.021 +1720 A3 26 A1 LSD0040.037 LLOQ1720 A3 18 A1 LSD0040.046 +1725 A3 26 A1 LSD0040.003 LLOQ1725 A3 40 A1 LSD0040.006 LLOQ1725 A3 26 A1 0.007 LLOQ1725 A3 18 A1 LSD0040.010 LLOQ1725 A3 40 A1 LSD0040.039 LLOQ1725 A3 18 A1 LSD0040.052 +1720 A3 403 A2 LSD0041.001 +1720 A3 399 A2 LSD0041.004 LLOQ1711 A3 403 A2 LSD0041.006 LLOQ1720 A3 403 A2 LSD0041.008 LLOQ1725 A3 403 A2 LSD0041.010 LLOQ1725 A3 403 A2 LSD0041.014 LLOQ1725 A3 399 A2 LSD0041.016 LLOQ1711 A3 403 A2 LSD0041.035 LLOQ1900 A3 399 A2 LSD0043.001 LLOQ1900 A3 403 A2 LSD0043.002 LLOQ1905 A3 403 A2 LSD0043.005 LLOQ1900 A3 399 A2 LSD0043.006 LLOQ1900 A3 403 A2 LSD0043.007 LLOQ1900 A3 403 A2 LSD0043.008 LLOQ1905 A3 399 A2 LSD0043.015 LLOQ1905 A3 403 A2 LSD0043.029 LLOQ1910 A3 403 A2 LSD0043.043 LLOQTable 25: Results of Coagulation Activity Assays for CFXTEN comprising three XTENInsertion 1 Insertion 2 Insertion 3ion . Insertion . Insertion . . .
. DomainSite Site.
Domain Domain uct ActiVitySite.
PCT/L S2012/046326hmmfionl Insertion 2 Insertion 3Insertion ADomain Insseitgon Domain Insortion Domain ConstructSite ActivitySite26 A1 403 A2 1656 A3 pSD0077 +++26 A1 403 A2 1720 A3 pSD0078 ++26 A1 403 A2 1900 A3 pSD0079 ++26 A1 1656 A3 1720 A3 pSD0080 +++26 A1 1656 A3 1900 A3 pSD0081 LLOQ26 A1 1720 A3 1900 A3 2403 A2 1656 A3 1720 A3 pSD0083 +++403 A2 1656 A3 1900 A3 pSD0084 +++403 A2 1720 A3 1900 A3 pSD00851656 A3 1720 A3 1900 A3 6 +++18 A1 745 B 2332 CT LSD0049.002 +++26 A1 745 B 2332 CT LSD0049.008 +++26 A1 745 B 2332 CT LSD0049.011 +++40 A1 745 B 2332 CT LSD0049.012 +++40 A1 745 B 2332 CT LSD0049.020 +++18 A1 745 B 2332 CT LSD0049.021 +++40 A1 745 B 2332 CT LSD0050.002 +++18 A1 745 B 2332 CT LSD0050.003 +++26 A1 745 B 2332 CT LSD0050.007 LLOQ18 A1 745 B 2332 CT LSD0050.010 +++26 A1 745 B 2332 CT LSD0050.012 +++40 A1 745 B 2332 CT LSD0050.014 +++403 A2 745 B 2332 CT LSD0051.002 +++399 A2 745 B 2332 CT LSD0051.003 +++403 A2 745 B 2332 CT LSD0052.001 +++399 A2 745 B 2332 CT LSD0052.003 +++1725 A3 745 B 2332 CT LSD0053.021 LLOQ1720 A3 745 B 2332 CT 3.022 +++1711 A3 745 B 2332 CT LSD0053.024 +++1720 A3 745 B 2332 CT LSD0054.021 +++1711 A3 745 B 2332 CT LSD0054.025 ++1725 A3 745 B 2332 CT LSD0054.026 +++1900 A3 745 B 2332 CT LSD0055.021 +++1905 A3 745 B 2332 CT LSD0055.022 +++1900 A3 745 B 2332 CT LSD0055.026 +++1900 A3 745 B 2332 CT LSD0056.021 +++1900 A3 745 B 2332 CT LSD0056.024 +++1910 A3 745 B 2332 CT LSD0056.025 +++0745 B 1900 A3 2332 CT pBC0294*0745 B 1900 A3 2332 CT *0745 B 1900 A3 2332 CT ch0296*0745 B 1900 A3 2332 CT *0745 B 1900 A3 2332 CT ch0298*0745 B 1900 A3 2332 CT ch0299*0745 B 1900 A3 2332 CT pBCO300*0745 B 1900 A3 2332 CT pBC0301*0745 B 1900 A3 2332 CT pBC0302*0745 B 1900 A3 2332 CT pBCO303*0745 B 1900 A3 2332 CT pBC0304*0745 B 1900 A3 2332 CT pBCO305*0745 B 1900 A3 2332 CT pBC0306*0745 B 1900 A3 2332 CT pBCO307* PCT/L S2012/046326Insertion 1 ion 2 Insertion 3Ingeitelon Domain Insseitgon Domain 3161011 Domain Construct Activity0745 B 1900 A3 2332 CT pBC0308*0745 B 1900 A3 2332 CT pBC0309*0745 B 1900 A3 2332 CT pBC0310*0745 B 1900 A3 2332 CT pBC0311*0745 B 1900 A3 2332 CT pBC0312*0745 B 1900 A3 2332 CT pBC0313*0745 B 1900 A3 2332 CT pBC0314*0745 B 1900 A3 2332 CT pBC0315*0745 B 1900 A3 2332 CT pBC0316*0745 B 1900 A3 2332 CT pBC0317*0745 B 1900 A3 2332 CT pBC0318*0745 B 1900 A3 2332 CT pBC0319*0745 B 1900 A3 2332 CT pBC0320*0018 A1 0745 B 2332 CT ch0269*0403 A2 0745 B 2332 CT ch0270*1720 A3 0745 B 2332 CT pBC0271*1900 A3 0745 B 2332 CT pBC0272*0403 A2 0745 B 2332 CT ch0273*1720 A3 0745 B 2332 CT 4*1900 A3 0745 B 2332 CT *0018 A1 0745 B 2332 CT ch0276*0403 A2 0745 B 2332 CT ch0277*1720 A3 0745 B 2332 CT ch0278*1900 A3 0745 B 2332 CT ch0279**Construct with R1648A mutationTable 26: Results of Coagulation Activity Assays for CFXTEN comprising four XTENXTEN XTEN XTEN XTEN XTEN XTENConstruct ID ActivityInsert 1 Insert 2 Insert 3 Insert 4 Insert 5 Insert 626 403 1656 1720 pSD008726 403 1656 1900 826 403 1720 1900 pSD0089 LLOQ26 1656 1720 1900 0403 1656 1720 1900 pSD00910040 0403 745 2332 ' LSD0058.006*0018 0409 745 2332 LSD0059.002*0040 0409 745 2332 LSD0059.006*0040 0409 745 2332 LSD0060.001*0018 0409 745 2332 LSD0060.003*0040 1720 745 2332 LSD0061.002*0026 1720 745 2332 LSD0061.007*0018 1720 745 2332 LSD0061.008*0018 1720 745 2332 LSD0061.012*0018 1720 745 2332 LSD0062.001*0026 1720 745 2332 LSD0062.002*0018 1720 745 2332 LSD0062.006*0018 1900 745 2332 LSD0063.001*0018 1900 745 2332 - - LSD0064.017*0026 1900 745 2332 - - LSD0064.020*0040 1900 745 2332 ' ' LSD0064.021*0040 1905 745 2332 ' ' LSD0065.001* --0018 1905 745 2332 - - LSD0065.014* --0040 1905 745 2332 - - LSD0066.001* --0026 1905 745 2332 ' ' LSD0066.002* --0018 1905 745 2332 - - LSD0066.009*0018 1905 745 2332 - - LSD0066.011*0018 1910 745 2332 ' ' LSD0067.004*0018 1910 745 2332 ' ' 7.005* --0040 1910 745 2332 - - LSD0067.006* --0026 1910 745 2332 - - LSD0067.008* --0018 1910 745 2332 ' ' LSD0068.001* --0026 1910 745 2332 - - LSD0068.002* --0040 1910 745 2332 - - LSD0068.005* --0018 1910 745 2332 - - LSD0068.010* ++0409 1720 745 2332 ' ' LSD0069.004* --0403 1720 745 2332 - - LSD0069.008* --0409 1720 745 2332 ' ' LSD0070.003* --0403 1720 745 2332 ' ' LSD0070.004*0403 1720 745 2332 - - LSD0070.005*0403 1900 745 2332 - - LSD0071.001*0403 1900 745 2332 - - LSD0071.002* +0409 1900 745 2332 - - LSD0071.008*0403 1900 745 2332 - - LSD0072.001*0403 1900 745 2332 ' ' LSD0072.002* --0409 1900 745 2332 ' ' 2.003* --0409 1905 745 2332 - - LSD0073.002* --0403 1905 745 2332 - - LSD0073.004* --0403 1905 745 2332 ' ' LSD0073.006* --0403 1905 745 2332 - - LSD0074.007* ++0409 1905 745 2332 - - LSD0074.010* --0403 1905 745 2332 ' ' LSD0074.011* --0409 1910 745 2332 ' ' LSD0075.004* --0403 1910 745 2332 - - LSD0075.007* --0403 1910 745 2332 - - LSD0076.002* --0403 1910 745 2332 ' ' LSD0076.003* --0403 1910 745 2332 - - 3* --1720 1900 745 2332 - - pSD0094* ++1720 1905 745 2332 ' ' pSD0095* --1720 1910 745 2332 ' ' pSD0097* --1720 1910 745 2332 - - 8* --0403 1656 1720 2332 ' ' pNL0022 --0403 1656 1900 2332 ' ' pNL0023 --0403 1720 1900 2332 ' ' pNL0024 LLOQ1656 1720 1900 2332 ' ' pNL0025 --0018 0403 1656 2332 ' ' pBC0247 ++0018 0403 1720 2332 ' ' pBC0248 --0018 0403 1900 2332 ' ' pBC0249 --0018 1656 1720 2332 ' ' pBC0250 --0018 1656 1900 2332 ' ' 10018 1720 1900 2332 ' ' 2 LLOQ0018 0403 0745 2332 ' ' LSD57.0050018 0745 1720 2332 ' ' LSD62.0010018 0745 1900 2332 ' ' pBC02620403 0745 1720 2332 ' ' LSD70.004 --0403 0745 1900 2332 ' ' pBC0266 --0745 1720 1900 2332 ' ' pBC0268 --0188 1900 0745 2332 - - pCS0001* ND0599 1900 0745 2332 - - pCS0002* ND2068 1900 0745 2332 - - pCS0003* ND2171 1900 0745 2332 - - pCS0004* ND2227 1900 0745 2332 - - pCS0005* ND2277 1900 0745 2332 - - pCS0006* ND0403 1656 1720 1900 2332 - pNL0030 LLOQ0018 0403 1656 1720 2332 --- 30018 0403 1656 1900 2332 --- pBC02540018 0403 1720 1900 2332 - pBC0255 LLOQ0018 1656 1720 1900 2332 --- pBC02560018 0403 0745 1720 2332 --- pBC0259*0018 0403 0745 1900 2332 --- pBC0260*0018 0745 1720 1900 2332 --- pBC02630403 0745 1720 1900 2332 - pBC0267 LLOQ0018 0403 1656 1720 1900 2332 pBC0257 LLOQ0018 0403 0745 1720 1900 2332 pBC0264 LLOQ*Construct with R1648A mutation Example 26: Determination ofXTEN Radii and related parameters In order to quantify the hydrodynamic radii of the XTEN components of CFXTEN fusionproteins and how the value of multiple XTEN versus single XTEN , a series of formulae werecreated based on empirically-derived data from size exclusion tography assays of various fusionproteins comprising one or more XTEN. It is ed that the incorporation of multiple XTEN into aCFXTEN provides a higher total hydrodynamic radius of the XTEN component compared to CFXTENwith fewer XTEN yet having approximately the same total ofXTEN amino acids. The maximum radiusWO 22617 of a single XTEN polypeptide is ated (hereinafter “XTEN Radius”) according to the formula givenby Equation 11:XTEN Radius = («/XTEN length 0.2037) + 3.4627 11 The sum of the maximum of the XTEN Radii for all XTEN segments in a CFXTEN iscalculated (hereinafter “Sum XTEN Radii”) ing to the formula given by Equation III:Z XTEN Radius1-i =1Sum XTEN Radii = 111wherein: n = the number ofXTEN segmentsand i is an or] The ratio of the SUM XTEN Radii of a CFXTEN comprising multiple XTEN to that of anXTEN Radius for a single XTEN of an equivalent length (in total amino acid residues to that of theCFXTEN) is calculated nafter “Ratio XTEN Radii”) according to the formula given by Equation[1.21 XTEN RadiusiRatio XTEN Radii = (1 ;;1XTEN Lengthg * 0.2037) + 3.4627wherein: n = the number ofXTEN segmentsand i is an iterator RLults: Equation 11 was d to XTEN of lengths 144, 288, 576 and 864. The results arepresented in Table 27. Equation IV was applied to various CFXTEN fusion proteins described hereinwith two, three, or four XTEN. The Ratio ofXTEN Radii has a value of l for all CFXTEN that contain asingle XTEN. The Ratio XTEN Radii are presented in Table 28. The Ratio ofXTEN Radii for pSDOO92,which contains 5 XTEN insertions, has a value of 3.31. Collectively, the results indicate that theinclusion of multiple XTEN increases the Ratio XTEN Radii to values greater than 2, with four insertionsresulting in higher values than three insertions.
Table 27: Results of Radii Calculations for CFXTEN comprising XTENXEN Len; XTEN RadiusTable 28: s of Radii Calculations for CFXTEN comprising XTENRatioInsert . Insert . InsertDomain Domain Domam. Insert XTENSite Site SiteSite Domain Construct Radii-_-_-_-_—_-——-—-——-——-—-—— PCT/U82012/046326Insertion 1 Insertion 2 Insertion 3 Insertion 4Ratio13:31 Insert InsertDomain Domam. Domain Insert XTENSite SiteSite Domain Construct Radii745 B 2332 CT 1.006 1.71745 B 2332 CT 1.011 1.71745 B 2332 CT LSD0001.012 1.71745 B 2332 CT LSD0001.013 1.67745 B 2332 CT 1.016 1.67745 B 2332 CT LSD0001.021 1.67745 B 2332 CT LSD0002.001 1.67745 B 2332 CT LSD0002.002 1.67745 B 2332 CT LSD0002.004 1.71745 B 2332 CT LSD0002.008 1.67745 B 2332 CT LSD0002.014 1.67745 B 2332 CT LSD0003.001 1.67745 B 2332 CT LSD0003.004 1.66745 B 2332 CT LSD0003.006 1.67745 B 2332 CT LSD0003.009 1.67745 B 2332 CT LSD0003.014 1.66745 B 2332 CT LSD0003.018 1.67745 B 2332 CT LSD0004.010 1.66745 B 2332 CT LSD0004.011 1.67745 B 2332 CT LSD0004.014 1.66745 B 2332 CT LSD0004.016 1.66745 B 2332 CT LSD0004.022 1.66745 B 2332 CT LSD0003.016 1.6726 A1 403 A2 LSD0005.002 1.7126 A1 403 A2 LSD0005.004 1.7140 A1 403 A2 LSD0005.005 1.7140 A1 403 A2 LSD0005.011 1.7118 A1 403 A2 LSD0005.018 1.7126 A1 599 A2 LSD0006.002 1.7140 A1 599 A2 LSD0006.005 1.7140 A1 599 A2 LSD0006.007 1.7140 A1 599 A2 LSD0006.011 1.7140 A1 403 A2 LSD0007.002 1.7140 A1 403 A2 LSD0007.004 1.7126 A1 403 A2 LSD0007.013 1.7126 A1 599 A2 LSD0008.001 1.7140 A1 599 A2 8.002 1.7126 A1 599 A2 LSD0008.006 1.7118 A1 599 A2 LSD0008.009 1.7140 A1 599 A2 LSD0008.017 1.71745 B 2332 CT LSD0002.025 1.71745 B 2332 CT LSD0002.013 1.67745 B 2332 CT LSD0003.025 1.67745 B 2332 CT 4.025 1.67745 B 2332 CT LSD0003.005 1.6626 A1 403 A2 LSD0007.008 1.711720 A3 1900 A3 LSD0044.002 1.711725 A3 1900 A3 LSD0044.005 1.711720 A3 1900 A3 LSD0044.039 1.71171 1 A3 1905 A3 LSD0044.022 1.711720 A3 1905 A3 LSD0044.003 1.71Insertion 1 Insertion 2 Insertion 3 Insertion 4Ratio13:31 Insert InsertDomain Domam. Domain Insert XTENSite SiteSite Domain uct Radii1725 A3 1905 A3 LSD0044.001 1.711656 A3 26 A1 LSD0038.001 1.711656 A3 18 A1 LSD003 8.003 1.711656 A3 18 A1 LSD0038.008 1.711656 A3 40 A1 LSD0038.012 1.711656 A3 40 A1 LSD0038.013 1.711656 A3 26 A1 LSD0038.015 1.711656 A3 399 A2 LSD0039.001 1.711656 A3 403 A2 LSD0039.003 1.711656 A3 403 A2 LSD0039.010 1.711656 A3 1725 A3 LSD0045.001 1.711656 A3 1720 A3 LSD0045.002 1.711900 A3 18 A1 LSD0042.014 1.711900 A3 18 A1 LSD0042.023 1.711900 A3 26 A1 LSD0042.006 1.711900 A3 26 A1 LSD0042.013 1.711900 A3 40 A1 LSD0042.001 1.711900 A3 40 A1 2.039 1.711900 A3 26 A1 LSD0042.047 1.711905 A3 18 A1 LSD0042.003 1.711905 A3 40 A1 LSD0042.004 1.711905 A3 26 A1 LSD0042.008 1.711905 A3 26 A1 LSD0042.038 1.711905 A3 40 A1 LSD0042.082 1.711910 A3 26 A1 2.040 1.7118 A1 399 A2 LSD0037.002 1.7126 A1 399 A2 LSD0037.009 1.7140 A1 399 A2 LSD0037.011 1.7118 A1 403 A2 LSD0047.002 1.7118 A1 403 A2 LSD0047.005 1.7118 A1 403 A2 LSD0048.007 1.711656 A3 1900 A3 LSD0046.001 1.711656 A3 1900 A3 LSD0046.002 1.711656 A3 1905 A3 LSD0046.003 1.711711 A3 40 A1 LSD0040.011 1.711711 A3 26 A1 LSD0040.042 1.711720 A3 26 A1 LSD0040.002 1.711720 A3 40 A1 LSD0040.008 1.711720 A3 18 A1 LSD0040.021 1.711720 A3 26 A1 LSD0040.037 1.711720 A3 18 A1 LSD0040.046 1.711725 A3 26 A1 LSD0040.003 1.711725 A3 40 A1 0.006 1.711725 A3 26 A1 0.007 1.711725 A3 18 A1 LSD0040.010 1.711725 A3 40 A1 LSD0040.039 1.711725 A3 18 A1 LSD0040.052 1.711720 A3 403 A2 LSD0041.001 1.711720 A3 399 A2 LSD0041.004 1.711711 A3 403 A2 LSD0041.006 1.711720 A3 403 A2 LSD0041.008 1.71Insertion 1 Insertion 2 Insertion 3 Insertion 4RatioInsert . Insert . InsertDomain Domaln Domaln.
Site Site Site InsertSlte . XTBNDomaln Construct Radn1725 A3 403 A2 LSD0041.010 1.711725 A3 403 A2 LSD0041.014 1.711725 A3 399 A2 LSD0041.016 1.711711 A3 403 A2 1.035 1.711900 A3 399 A2 LSD0043.001 1.711900 A3 403 A2 LSD0043.002 1.711905 A3 403 A2 LSD0043.005 1.711900 A3 399 A2 LSD0043.006 1.711900 A3 403 A2 LSD0043.007 1.711900 A3 403 A2 LSD0043.008 1.711905 A3 399 A2 LSD0043.015 1.711905 A3 403 A2 3.029 1.711910 A3 403 A2 LSD0043.043 1.7126 A1 403 A2 1656 A3 pSD0077 2.3026 A1 403 A2 1720 A3 pSD0078 2.3026 A1 403 A2 1900 A3 pSD0079 2.3026 A1 1656 A3 1720 A3 pSD0080 2.3026 A1 1656 A3 1900 A3 pSD0081 2.3026 A1 1720 A3 1900 A3 pSD0082 2.30403 A2 1656 A3 1720 A3 pSD0083 2.30403 A2 1656 A3 1900 A3 pSD0084 2.30403 A2 1720 A3 1900 A3 pSD0085 2.301656 A3 1720 A3 1900 A3 pSD0086 2.3026 A1 403 A2 1656 A3 1720 A3 pSD0087 2.8326 A1 403 A2 1656 A3 1900 A3 pSD0088 2.8326 A1 403 A2 1720 A3 1900 A3 pSD0089 2.8326 A1 1656 A3 1720 A3 1900 A3 pSD0090 2.83403 A2 1656 A3 1720 A3 1900 A3 pSD0091 2.8326 A1 403 A2 1656 A3 1720 A3 pSD0092 2.8318 A1 745 B 2332 CT 9.002 2.2426 A1 745 B 2332 CT LSD0049.008 2.2426 A1 745 B 2332 CT LSD0049.011 2.2440 A1 745 B 2332 CT LSD0049.012 2.2440 A1 745 B 2332 CT LSD0049.020 2.2418 A1 745 B 2332 CT LSD0049.021 2.2440 A1 745 B 2332 CT 0.002 2.2418 A1 745 B 2332 CT LSD0050.003 2.2426 A1 745 B 2332 CT LSD0050.007 2.2418 A1 745 B 2332 CT LSD0050.010 2.2426 A1 745 B 2332 CT LSD0050.012 2.2440 A1 745 B 2332 CT LSD0050.014 2.24403 A2 745 B 2332 CT LSD0051.002 2.24399 A2 745 B 2332 CT LSD0051.003 2.24403 A2 745 B 2332 CT LSD0052.001 2.24399 A2 745 B 2332 CT LSD0052.003 2.241725 A3 745 B 2332 CT LSD0053.021 2.241720 A3 745 B 2332 CT LSD0053.022 2.241711 A3 745 B 2332 CT LSD0053.024 2.241720 A3 745 B 2332 CT LSD0054.021 2.241711 A3 745 B 2332 CT 4.025 2.241725 A3 745 B 2332 CT LSD0054.026 2.24Insertion 1 Insertion 2 Insertion 3 Insertion 4RatioInsertInsertSite . XTENDomain Construct Radn1900 B1905 B1900 B1900 B1900 B1910 B Exam le 27: Bindin Interference of XTEN t0 anti-FVIII Antibod The y ofXTEN inserted into different ons of CFXTEN fusion proteins to affect thebinding of VIII dies was determined by ch ELISA assays. Two anti-FVIII antibodies;i.e. GMA—8021 (Green Mountain Antibodies, Burlington, VT) and ESH8 (American Diagnostica Inc.,Stamford, CT), that bind to the A2 and C2 s, respectively were ed as capture antibodies. Anon-XTEN containing FVIII-His-Myc protein was used as a calibration standard and positive control forall ELISAs. Ten CFXTEN fusion proteins with single XTEN insertions in either the Al, A2 or A3domains were created that onally contained His and Myc affinity tags. The protein concentrationsof each test sample was normalized to 100% based on an anti-His capture-anti-Myc detection ELISA runconcurrently on the same plate as the anti-FVIII antibody capture-anti-Myc detection ELISA.
Briefly, appropriate wells on a 96-well plate were coated with GMA—8021, ESH8 or anti-Hisantibody ght at 4°C, then were washed and blocked with BSA. Equal volumes of the respectivecontrol or fusion proteins were introduced into ate wells and allowed to interact with coated GMA-8021, ESH8 or anti-His antibody for 2h at room ature. After incubation, d material waswashed away and a rabbit anti-Myc detection antibody was added and incubated for an additional h atroom temperature. The plate was then washed and a peroxidase-conjugated donkey anti-rabbit secondaryantibody was introduced and incubated for 1h at room temperature. The plate was washed again,followed by the addition of TMB substrate and the reaction was allowed to proceed for 5-20 min.
H2SO4 was introduced to stop the reaction and absorbance was read by spectrophotometer at 450nm.
RLults: The results are presented in Table 29. Collectively, the results demonstrate that thetwo antibodies against the CFXTEN fusion proteins with XTEN inserted into the A2 domain exhibitedreduced binding of FVIII ed to CFXTEN with XTEN inserted into the Al or A3 domain when theanti-FVIII capture antibody was GMA—8021 (with binding affinity to the A2 domain). In contrast, therewas no discernible pattern of inhibition or enhancement of binding by any of the CFXTEN when the anti-FVIII capture antibody was ESH8, with binding affinity to the C2 domain.
Table 29: Bindin Interference of XTEN t0 anti-FVIII AntibodXTEN insertion Concentration on aFVIII/Myc + tration on aHis/MycSample Tested (Domain, site,His/M GMA—SOZl/Myc (A2 ESHS/MycXTEN) ycdomain) ((12 domain)FVIII-His-Myc 100% 104%FVIII-XTEN—His- A2, 403, AE144 100% 103%: 1% 141%:24%Myc A2, 403, AG144 100% 104% :: 6% 129% :: 12%A2, 399, AE144 100% 100% :: 8% 140% :: 18%A3, 1656, AG144 100% 153% 158%A1, 18, AE144 100% 129% 130%A1,18, AG144 100% 150% 131%A1, 26, AE144 100% 155% 87%A1, 26, AG144 100% 157% 147%A1, 40, AE144 100% 137% 147%A1, 40, AG144 100% 164% :: 0% 153% :: 18%aFVIII/Myc = GMA—8021/Myc or ESH8/Myc antibody condition; aHis/Myc = anti-His/Myc antibody Example 28: Activity Assay of CFXTEN fusion proteins in the presence of FVIIIinhibitors Inhibitor Testing Titration Procedure: Select antibodies ting FVIII procoagulant activity were purchased from cialsources. The antibodies target select domains of FVIII (e. g. A2, A3, C1, C2) and inhibit FVIII-dependent gulant ty. In order to establish the l concentration of FVIII inhibitors toutilize in the assay, an initial titration experiment was performed using varying amounts of eachinhibitory antibody incubated at 37°C for 2 hrs with the base vector sing wild-type FVIII with aHis/Myc double tag, and a second sample with antibody and at least one CFXTEN fusion protein. Thesamples were then utilized in a coagulation assay to determine the FVIII ty. The activity wasmeasured by the Coatest assay procedure described . The concentration that resulted in optimaltion of FVIII activity was determined for each antibody individually.
Inhibitor Testing Procedure: The FVIII tor antibodies were then used at their optimal concentration for assay of testsamples. CFXTEN and positive control samples were individually incubated with each antibody at 37°Cfor 2 hrs and the samples were then collected and utilized in the Coatest activity assay, along withuntreated aliquots of the CFXTEN and positive control. In some cases, CFXTEN constructs with aR1648A mutation were tested to determine the effect, if any, of this mutation on resistance to inhibitorsas measured by the retention of FVIII activity.
] Res—ults: The results of the titration experiment are shown in . The data indicate a right-shift ofapproximately 0.7 order of magnitude in the amount of antibody required to inhibit the procoagulantactivity of the CFXTEN LSD0049.002 to the 50% level, compared to FVIII positive control, indicatingthat the CFXTEN with three XTEN insertions (at ion points corresponding to amino acid residue18, 745 and 2332 of the BDD-FVIII) had lower binding with the antibody compared to FVIII, reflectedin the retention of coagulation activity.
] The results of the Coatest assays are ted in Tables 30 and 31, for the FVIII inhibitorantibodies GMA8008 and GMA8021, respectively. All of the untreated CFXTEN fusion proteinconstructs tested exhibited procoagulant activity, as did the pBCOOl 14 FVIII positive control. Thepositive control sample pre-incubated with FVIH inhibitor antibodies resulted in a sharp decrease in themeasured coagulation activity to 0.05-O.15 (5-15%) relative to the untreated sample, as did the majorityof the CFXTEN constructs treated with the GMA8008 antibody to the C2 . However, threeCFXTEN fusion proteins retained at least twice the relative remaining activity compared to the FVIIIcontrol; LSDOO49.020, LSD0053.024, and LSD0056.025, each with three XTEN inserts.
The CFXTEN s showed a lower degree of inhibition with the GMA8021 antibody to theA2 domain ed to untreated s that was further reduced by either the additional numbers ofXTEN inserts (tabular data shown in Table 30). shows the graph of median values of the ratio tocontrol of retained activity g a linear relationship between numbers ofXTEN ed and reducedinhibition to the GMA8021 antibody ve to the inhibition of the FVIII control. Similarly, the meansi S.E. for the ratio to control values were 2.26i0.l2 for l XTEN, 3.48+O.26 for 2 XTEN and 5.70i0.29for 3 XTEN insertions. CFTXEN with at least three XTEN inserts treated with the GMA8021 antibodyhad at least 4.5 to 9.2-fold greater retention of FVIII activity compared to FVIII control. In addition, inthose CFXTEN with three XTEN ions, constructs with a higher degree of separation (in numbers ofamino acid residues) between any two insertions appeared to result in a higher degree of procoagulantactivity and, hence, less binding by the FVIH inhibitor antibody, compared to insertions red moreclosely; e.g. on the C-terminal side of the B-domain. The assay results of constructs with the R1648Amutation appeared to be comparable to those t the mutation.
Conclusions: The results support that, under the conditions of the experiments, insertion ofXTEN into FVIH resulted in protection against binding by FVIII inhibitors, with retention ofprocoagulant activity, and that inclusion of multiple XTEN inserts increased ance to, in particular,the A2 domain tor antibody. Lastly, there appears to be an effect by having spatial tionbetween the XTEN inserts.
Table 30: Results of Coagulation Assay with CFXTEN treated with antibody GMA8008 to C2DomainConstruct RelaF‘Y" Ratio t0 XTEN XTEN XTENRemaining .. . . onsNamel Insertlon 1 Insertion 2 Insertion 3pBC0114 CT 0.05-0.15 lpBC0149 0.1 0.8 0745_AE42_10018_AE144_5pSD0045 0.3 l . l ApSD0046 0.3 1.0 0018_AG144_FpSD0050 0.2 0.9 0026_AG144_F0040_AE144_5pSD0051 0.3 1.3 ApSD0052 0.2 1.0 0040_AG144_F0403_AE144_2pSD0001 0.2 0.9 ApBC0136 0.2 1.2 0745_AE288_17 0.2 1.1 0745_AE288_1 R1648AW0 2013/122617 2012/046326Construct Relarwe Ratio to XTEN XTEN XTENRemalnmg .
Name Control Insertlon 1. Insertion 2. . Mutatlons. Insertion 3Actmty.pSD0013 0.1 0.9 2332_AE144_6BpSD0014 0.1 0.8 G144_1BC0145 0.1 0.6 2332 AE288 1pSD0019 0.1 0.5 E288_1pBC0146 0.1 0.7 2332_AG288_1pSD0015 0.1 0.8 2332_AE8648.008 0.1 0.9 0018_AG144_F 1656_AG144_CLSD003 8.013 0.1 0.6 0040_AG144_F 1656_AG144_CLSD003.09 0.1 0.9 0745_AE144_3B 2332_AE288_1LSD003.06 0.0 0.8 0745_AE144_3B 2332_AE288_1 R1648ALSD0046.001 0.0 0.6 1656_AG144_C 1900_AG144_C0403_AE144_2 1656_AG144_PSD077 0.1 1.0 G144_F A C1720_AG144_PSD080 0.1 1.0 G144_F 1656_AG144_C C0403_AE144_2 1720_AG144_PSD083 0.1 0.8 A 1656_AG144_C CE144_2 1900_AE144_PSD084 0.1 0.9 A 1656_AG144_C 4A0018_AE144_5 2332_AE288_LSD0050.010 0.1 0.7 A 0745_AE144_3B 10018_AE144_5 2332_AE288_LSD0049.021 0.0 0.6 A 0745_AE144_3B 1 R1648A2332_AE288_LSD0049.002 0.1 0.9 0018 AG144 F 0745 AE144 3B 1 R1648A0026_AE144_5 2332_AE288_LSD0049.008 0.1 0.9 A 0745_AE144_3B 1 R1648A2332_AE288_LSD0049.011 0.1 0.9 0026_AG144_F 0745_AE144_3B 1 R1648A0040_AE144_5 E288_LSD0049.020 0.2 2.6 A 0745_AE144_3B 1 R1648A2332_AE288_LSD0050.002 0.0 0.2 G144_F 0745_AE144_3B 11711_AE144_4 2332_AE288_LSD0053.024 0.2 2.5 A 0745_AE144_3B 12332_AE288_LSD0054.021 0.2 1.5 1720_AG144_C 0745_AE144_3B 11900_AE144_4 E288_LSD0055.021 0.2 1.6 A 0745_AE144_3B 1 R1648A2332_AE288_LSD0056.021 0.2 1.6 1900_AG144_C 0745_AE144_3B 12332_AE288_LSD0056.025 0.3 2.0 1910_AG144_C 0745_AE144_3B 1proportion of activity remaining relative to corresponding untreated sampleThe ratio of the relative remaining activity (relative to its own control) compared to FVIII pBC0114positive controlTable 31: Results of Coagulation Assay with CFXTEN treated with antibody GMA8021 to A2DomainAetiVity ControlpBC0114 0.05-0.15 1pBCO 149 0.2 1.3 0745_AE42_1pSD0045 0.3 2.7 0018_AE144_5ApSD0046 0.2 2.1 0018_AG144_FpSD0050 0.2 2.4 0026_AG144_FpSD0051 0.3 3.1 0040_AE144_5ApSD0052 0.3 2.7 0040_AG144_FpSD0001 0.2 1.6 0403 AE144 2ApBC0136 0.3 2.4 0745_AE288_1pBC0137 0.3 2.4 0745_AE288_1 R1648ApSD0013 0.2 1.8 2332_AE144_6BpSD0014 0.2 2.1 2332_AG144_1pBC0145 0.3 2.1 2332_AE288_1pSD0019 0.3 2.3 2332_AE288_1pBC0146 0.3 2.1 2332_AG288_1pSD0015 0.3 2.8 2332_AE864LSD0038.008 0.4 3.0 0018_AG144_F 1656_AG144_CLSD0038.013 0.4 3.0 0040_AG144_F 1656_AG144_CLSD003.09 0.3 3.6 0745_AE144_3B E288_1LSD003.06 0.3 3.4 0745 AE144 3B 2332 AE288 1 R1648A6.001 0.2 4.4 G144_C 1900_AG144_CPSD077 0.4 5.8 0026_AG144_F 0403_AE144_2A 1656_AG144_CPSD080 0.4 5.7 0026_AG144_F 1656_AG144_C 1720_AG144_CPSD083 0.3 5.0 0403_AE144_2A 1656_AG144_C 1720_AG144_CPSD084 0.3 4.5 E144_2A 1656_AG144_C 1900_AE144_4ALSD0050.010 0.4 6.7 0018_AE144_5A 0745_AE144_3B 2332_AE288_1LSD0049.021 0.4 6.7 E144_5A 0745_AE144_3B 2332_AE288_1 R1648A9.002 0.5 9.2 0018_AG144_F 0745_AE144_3B 2332_AE288_1 R1648ALSD0049.008 0.4 5.9 0026_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648ALSD0049.011 0.4 5.6 0026_AG144_F 0745_AE144_3B 2332_AE288_1 R1648ALSD0049.020 0.3 5.0 0040_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A0.002 0.3 6.2 0040 AG144 F 0745 AE144 3B 2332 AE288 1LSD0053 .024 0.3 4.5 1711_AE144_4A E144_3B 2332_AE288_1LSD0054.021 0.5 5.2 1720_AG144_C 0745_AE144_3B E288_1LSD0055.021 0.5 5.4 E144_4A 0745_AE144_3B 2332_AE288_1 R1648ALSD0056.021 0.5 5.1 1900_AG144_C 0745_AE144_3B 2332_AE288_1LSD0056.025 0.5 4.8 1910_AG144_C 0745_AE144_3B 2332_AE288_1proportion of activity ing relative to corresponding untreated sampleThe ratio of the relative remaining activity (relative to its own control) compared to FVlll 4positive control Example 29: Protein ation of CFXTEN fusion proteins pBC0145 and pBC0146 Two CFXTEN constructs with C-terminal XTEN were utilized to ish a purificationmethod. For both pBC0145 with a C-terminal XTEN of 288 amino acids of the AE family (see sequencein Table 21) and pBC0146 with a C-terminal XTEN of 288 amino acids of the AG family (see sequencein Table 21), a tial flow filtration (TFF) step was used to buffer ge the ed conditionedmedia from cell culture. Products were then captured using a strong anion exchange chromatographyresin, and then further purified using VIIIS elect affinity chromatography (GE care). An additionalsize exclusion tography (GE Healthcare) was applied to FVIII-pBC0146 as a third polish step toremove high molecule weight species. The purity of both fusion proteins was deemed acceptable byHPLC-SEC and was further confirmed by SDS-PAGE analysis of the two CFXTEN ucts showingCFXTEN products at expected sizes. The specific ty of both molecules was comparable to B-domain deleted FVIII, as measured by aPTT coagulation assay and ELISA determination of FVIIIconcentration.
Example 30: Pharmacokinetics of CFXTEN fusion proteins 5 and pBC0146 inHemA and FVIIINWF DKO mice Male FVIII knock-out (HemA) mice or VWF double knock-out (DKO) mice, 8-12weeks old, were d with a single intravenous administration of either recombinant BDD-FVIII, theCFXTEN pBC0145 0r pBC0146 fusion purified proteins (from Example 23) at 200 IU/kg dose (n=4/timepoint). At select time points, blood samples were collected via vena cava sampling. In HemA mice,blood samples were collected at 5 min, 1 4, 8, 16, 20, 24, 32, and 48 hrs post-dosing for rBDD-FVIII,and at 5 min, 8, 16, 24, 32, 48, 55 and 72 hrs post-dosing for pBC0145 and pBC0146 fusion proteins. Inthe FVIII/VWF DKO mice, blood samples were collected at 5min, 30 min and 1hr osing forrBDD-FVIII, and at 5 min, 4, 8, 16 and 24 hr post-dosing for the pBC0145 and pBC0146 fusion proteins.
Plasma FVIII activity was measured by FVIII chromogenic assay and the PK profile was analyzed by theWinNonlin program.
Res—ults: As show in Table 32 and , CFXTEN with the AE C-terminus XTEN insertion(pBC0145) ted 1.6-fold and old FVIII half-life (Tl/2) extension compared to rBDD FVIII inHemA mice and FVIII/VWF DKO mice, respectively. The CFXTEN with the AG C-terminus XTENinsertion (pBC0146) had 1.4-fold and 14.4-fold extended half-life compared to rBDD-FVIII in the HemAmice and FVIII/VWF DKO mice, respectively. The magnitude of the FVIII half-life extension conferredby XTEN insertion was much more pronounced in the FVIII/VWF DKO mice compared to the HemAmice, demonstrated by the 14-fold longer FVIII half-life from both FVIII-AE-XTEN and FVIII-AG-XTEN compared to rBDD-FVIII. In addition, in comparison to VIII, FVIII with C-terminal AE0r AG-XTEN insertion also had significantly improved FVIII recovery at the 5 min al, reducedclearance and volume of distribution, and increased AUC in the DKO mice. Under the conditions of theexperiment, CFXTEN with C-terminus XTEN insertions demonstrated great potential on FVIII half-lifeion, and, when combined with other FVIII intra-domain insertions could potentially r extendFVIII half-life.
Table 32: cokinetic parameters of CFXTEN in HemA and FVIIINWF DKO micemin AUC DMouse Tm MRT C1 Vss — Tm Fold Mouse. Treatment Recovery (hrkngU/mStrain (hr) (hr) (mL/hr/kg) (mL/kg) Increase Strain.
(%) L/mIU)73 11.88 16.47 3.81 62.74 0.26 1.6 HemApBC0146 64 10.54 13.31 5.66 75.34 0.18 1.4HemAr3731]; 89 7.58 11.02 4.33 47.68 0.23FVIII/pBC0145 74 3.38 3.76 13.06 63.68 0.0765 13.9 VWFFVIII/pBC0146 61 3.45 3.61 17.40 86.63 0.0575 14.2r3731]; 23 0.24 0.24 460.62 161.51 0.0022Compared to rBDD-FVIII Example 31: Cell e and concentration of cell culture media for CFXTEN fusionproteins pSD0050 and pSD0062 CFXTEN construct variants pSD0050 with an intradomain AG XTEN of 144 amino acidsinserted after amino acid residue 26 of BDD FVIII with an omain AE XTEN of 144, pSDOO62amino acids inserted after residue 1900 of BDD FVIII (Note: amino acid numbering based full lengthFVIII), as well as a construct encoding rBDD-FVIII, were transfected into HEK293F cells (Invitrogen,Carlsbad, CA) using polyethyleneimine (PEI, Polysciences Inc. Warrington, PA). The transientlytransfected cells were grown in 293 Free Style medium media (Invitrogen, ad, CA) for 4 days and50-100 ml cell culture media were then concentrated 10- to 20-fold by Centricon Spin Column (100 kDaMW cut-oft) to reach 10-30 IU/ml FVIII activity. The concentrated materials were then flash-frozen andstored at -80°c for future in vitro analysis and in vivo pharmacokinetic studies.] e 32: Pharmacokinetics of CFXTEN fusion proteins pSD0050 and pSD0062 inHemA and FVIIINWF DKO mice Male HemA or FVIII/VWF double knock-out (DKO) mice, 8-12 weeks old, were treated witha single intravenous administration of cell culture concentrates from Example 31 containing eitherrecombinant BDD-FVIII, the CFXTEN O or pBD062 at 100-300 IU/kg (n=3/group). At selecttime points, blood samples were collected Via retro orbital bleeds from the same set of mice. In HemAmice, blood samples were collected at 5 min, 24 hr and 48 hr post-dosing, while in VWF DKOmice blood samples were collected at 5 min, 8 hr and 16 hr. The FVIII activity of plasma samples andcell culture concentrates were analyzed by a FVIII chromogenic assay, and the PK profile of rBDD FVIIIand FVIII-XTEN variants were analyzed using the WinNonlin program.
] Res—ults: The PK profiles of the two CFXTEN intradomain insertion variants pSD0050 andpSDOO62 and rBDD-FVIII in HemA mice and FVIII/VWF DKO mice are shown in and Table33. In HemA mice, a comparable initial recovery at the 5 min interval was observed for the three testFVIII molecules. Both CFXTEN fusion proteins demonstrated two-fold longer half-life compared towild-type III. In FVIII-VWF DKO mice, because of the loss ofVWF protection, rBDD-FVIIIhad only a 15 min plasma half-life. In the case of the two CFXTEN, however, ife were extended to3.15 hr and 3.83 hr, respectively; values that are comparable to the CFXTEN with 288 C-terminus XTENions (Example 24), suggesting that further extension of the XTEN length at a given insertion pointmay not be necessary. Under the experimental conditions, the study s clearly trate thatintradomain insertion of an XTEN with 144 amino acid residues not only preserved FVIII ty, butalso provided r FVIII half-life benefit as the C-terminus 288 amino acid XTEN insertion variants,suggesting that the combination of the FVIII intradomain and C-terminus ions may allow rextension of FVIII half-life.
Table 33: Pharmacokinetic parameters of CFXTEN in HemA and FVIIINWF DKO miceMouseTreatment Rejcbriigry Tm MRT Cl VSS (hrfgtlrrcilfi?mL Tm POMStram (hr) (hr) (mL/hI/kg) (mL/kg) Increase(%) /mIU)pSD0050 40 14.12 14.25 5.27 75.03 0.19 2.3HemA pSD0062 43 12.96 14.79 4.24 62.67 0.24 2.1Tl?‘1?11131_ 47 6.19 2.62 6.35 16.62 0.16pSD0050 34 3.15 2.59 21.73 56.28 0.05 ~12FVIII/3.83 3.71 18.51 68.69 0.05 ~15VWF pSD0062DKO T331? 23 ~0.25Compared to rBDD-FVIII Example 33: Pharmacokinetic analysis of CFXTEN fusion polypeptides in rats The pharmacokinetics of various CFXTEN fusion ns, compared to FVIII alone, are testedin Sprague-Dawley rats. CFXTEN and FVIII are administered to female Sprague-Dawley rats (n=3) IVthrough a r vein catheter at 3-10 ug/rat. Blood samples (0.2 mL) are collected into pre-chilledheparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and processed into plasma.
Quantitation of the test articles is performed by ELISA assay using an anti-FVIII antibody for bothcapture and detection. A non-compartmental analysis is performed in WinNonLin with all time pointsincluded in the fit to determine the PK ters. Results are expected to show increased terminal half-life and area under the curve, and a reduced volume of distribution for the CFXEN compared to FVIIIalone, and the results are used in conjunction with results from coagulation and pharmacodynamic assaysto select those fusion protein configurations with desired properties.
Example 34: Analysis of FVIII for XTEN insertion sitesThe selection ofXTEN ion sites within the factor VIII molecule was performed by predicting thelocations of permissive sites within loop ures or otherwise flexible surface exposed structuralelements. For these analyses, the atomic coordinates of two independently determined X-raycrystallographic structures of FVIII were use (Shen BW, et al. The tertiary structure and domainorganization of coagulation factor VIII. Blood. (2008) Feb 1;111(3):1240-1247; Ngo JC, et al. lstructure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex.
Structure (2008)16(4):597-606), as well as those of factor VIII and factor VIIIa derived from moleculardynamic simulation (MDS) (Venkateswarlu, D. Structural investigation of zymogenic and activatedforms of human blood coagulation factor VIII: a ational molecular dynamics study. BMC StructBiol. (2010) 10:7). Atomic coordinates in Protein Data Bank (PDB) format were ed to identifyregions of the FVIII/FVIIIa predicted to have a high degree solvent accessible e area using thealgorithms ASAView (Ahmad S, et al. ASAView: database and tool for t accessibilityentation in proteins. BMC Bioinformatics (2004) 5:51) and GetArea (Rychkov G, Petukhov M.
Joint neighbors imation of macromolecular solvent accessible surface area. J Comput Chem(2007) 28(12):1974-1989). The resulting set of sites was then further prioritized on the basis of highpredicted atomic positional fluctuation based on the basis of the published results of the MDS study.
Sites within the acidic peptide regions flanking the A1, A2, and A3 domains, as well as those thatappeared by visual inspection to be in areas other than surface exposed loops were deprioritized. Theresulting set of ial sites was evaluated on the basis of pecies sequence conservation, withthose sites in regions of high sequence conservation among 20 vertebrate species being ranked morefavorably. Additionally, putative clearance receptor binding sites, FVIII interaction sites with othermolecules (such as vWF, FIX), domain and exon boundaries were also considered in fusion siteselection. Finally, sites within close proximity to mutations implicated in ilia A listed in theHaemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database were ated(Kemball-Cook G, et al. The factor VIII Structure and on Resource Site: HAMSTeRS version 4.
Nucleic Acids Res. (1998) 26(1):216-219). Based on these criteria, the construction of 42 FVIII-XTENvariants was proposed for XTEN insertions. Of these, three represent XTEN insertions within theresidual B domain sequence, two represent ions to the C-terminus of the factor VIII molecule, and37 ent XTEN insertions within structurally def1nedinter- and intradomain structural elements; i.e.,residues 3, 18, 22, 26, 40, 60, 116, 130, 188, 216, 230, 333, 375, 403, 442, 490, 518, 599, 713, 745, 1720,1796,1802,1827, 1861,1896, 1900,1904,1937, 2019, 2068, 2111, 2120, 2171, 2188, 2227, 2277, and2332.
Example 35: Functional analysis of FVIII-XTEN constructs Two FVIII-XTEN fusion proteins, FVIII-AE288 0) and FVIII-AG288 (F8X-41),contain an AE288_1 XTEN or an AG288_1 XTEN, respectively, fused at the inus of FVIII C2domain. To determine if FVIII ty was retained after XTEN fusion, HEK293 cells were transfectedseparately with these two FVIII-XTEN fusion constructs by using polyethylenimine (PEI) in serum-freemedium. At 3 or 5 days post-transfection, the cell culture supernatant was tested for FVIII activity by atwo-stage chromogenic assay. Purified recombinant FVIII, calibrated against WHO internationalstandard, was used to establish the standard curve in the geinic assay. The fusion proteinproducts of both F8X-40 and F8X-41 constructs were expressed at levels comparable to those of wild-type BDD-FVIII constructs. (Table 34).
PCT/U82012/046326Table 34. FVIII Titer of FVIII-XTEN fusion roteins in transient transfection cell cultureFVIII Molecules FVIII O66a pBC 0114a F8X~40 F8X~41awn Ba. Both FVIII 066 and pBC 0114 contain B-domain deleted FVIII without XTEN .b. The F8X-4lsample was from a 3-day transfection while other samples were from a 5-day transienttransfection.
Example 36: Functional is of FVIII-XTEN constructs: FVIII activity and PKproperties The ife extension ial of the F8X-4O and F8X-4l constructs was evaluated in FVIIIand von Willebrand factor double knock-out mice by hydrodynamic plasmid DNA injection, with aFVIIIFc DNA construct serving as a ve control. Mice were randomly divided into 3 groups with 4mice per group. Plasmid DNA encoding BDD c fusion protein, F8X-4O or F8X-4l, all sharing thesame DNA vector backbone, was administered to mice in the respective groups. Approximately 100micrograms of the appropriate plasmid DNA was injected into each mouse via hydrodynamic injection,and blood plasma samples were collected at 24 hours and 48 hours post-injection. The plasma FVIIIactivity was measured by a two-stage chromogenic assay using calibrated recombinant FVIII as astandard. As shown in , samples from the F8X-4O and F8X-4l groups showed higher plasmaFVIII titers than did those from the BDD FVIIIFC, suggesting FVIII fusion with XTEN prolongs the half-life of FVIII in viva. Taken together, these data support the conclusion that XTEN fusion proteinsretained FVIII activity in transient transfection and exhibited prolonged ating half-life in an animalmodel.
Example 37: Pharmacodynamic evaluation of CFXTEN in animal models The in vivo pharmacologic activity of CFXTEN fusion proteins are ed using a varietyof nical models of bleeding ing but not limited to those of hemophilia, surgery, trauma,ocytopenia/platelet ction, clopidogrel/heparin—induced bleeding and hydrodynamicinjection. These models are developed in multiple species including mice, rat, rabbits, and dogs usingmethods equivalent to those used and published for other FVIII approaches. CFXTEN compositions areprovided in an aqueous buffer compatible with in vivo administration (for example: phosphate-bufferedsaline or Tris-buffered saline). The compositions are administered at appropriate doses, dosingfrequency, dosing schedule and route of administration as optimized for the particular model. Efficacydeterminations e measurement of FVIII activity, one-stage clotting assay, FVIII chromogenicassay, activated partial prothrombin time (aPTT), bleeding time, whole blood clotting time (WBCT),thrombelastography (TEG or ROTEM), among others.
In one example of a PD model, CFXTEN and FVIII are administered to genetically-deficientor experimentally-induced HemA mice. At various time points post-administration, levels of FVIII andCFXTEN are measured by ELISA, activity of FVIII and CFXTEN is ed by commercially-available FVIII ty kits and clotting time is measured by aPTT assay. l, the results canindicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as ed toFVIII and/or equivalent in y to comparable dosage of FVIII with less frequent or more convenientdosing intervals.
In a mouse bleeding challenge PD model CFXTEN and FVIII are administered to genetically-deficient or experimentally-induced HemA mice and effect on hemostatic challenge is ed.atic challenge can include tail transaction challenge, hemarthropthy challenge, joint bleeding orsaphenous vein nge among . At various time points post-administration levels of FVIII andCFXTEN are measured by ELISA, activity of FVIII and CFXTEN are measured by commerciallyavailable FVIII activity kit, bleeding time is measured and clotting time is measured by aPTT assay.
Overall the results are expected to indicate that the CFXTEN constructs are more ious at inhibitingbleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII With lessfrequent or more convenient dosing intervals, and the results are used in conjunction With results fromcoagulation and other assays to select those fusion protein configurations With desired properties.
In a dog PD model, CFXTEN and FVIII are administered to genetically-deficient hemophiliacdogs. At various time points post administration, levels of FVIII and CFXTEN are measured by ELISA,activity of FVIII and CFXTEN are measured by commercially available FVIII activity kit and clottingtime is measured by aPTT assay. Overall the results indicates that the CFXTEN constructs may be moreefficacious at inhibiting bleeding as compared to FVIII and/or equivalent in y to abledosage of FVIII With less frequent or more convenient dosing, and the results are used in conjunctionWith results from coagulation and other assays to select those fusion protein configurations With desiredproperties.
In a dog bleeding challenge PD model CFXTEN and FVIII are administered to geneticallydeficient hemophiliac dogs and effect on hemostatic challenge is measured. atic challengeincludes cuticle bleeding time among others. At various time points post-administration levels of FVIIIand CFXTEN are ed by ELISA, activity of FVIII and CFXTEN are measured by commerciallyavailable FVIII activity kit, bleeding time is measured and clotting time are measured by aPTT assay.
Overall the results indicate that the CFXTEN constructs may be more efficacious at ting bleedingas compared to FVIII and/or equivalent in potency to comparable dosage of FVIII With less frequent ormore convenient dosing intervals, and the results are used in conjunction With results from coagulationand other assays to select those fusion protein configurations With desired properties.
] Additional preclinical models of bleeding include but are not limited to those of hemophilia,surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin—induced bleeding andynamic ion. These models can developed in multiple species including mice, rat, rabbits,and dogs using methods equivalent to those used and published for other FVIII approaches. Overall theresults indicate that the CFXTEN ucts may be more efficacious at inhibiting bleeding as comparedto FVIII and/or equivalent in potency to able dosage of FVIII with less frequent or moreient dosing intervals, and the results are used in conjunction With results from coagulation andother assays to select those l protein configurations With d properties.
PCT/U82012/046326 Example 38: CFXTEN with cleavage sequences C-terminal XTEN releasable by FXIa A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII iscreated with an XTEN release site cleavage ce placed in between the FVIII and XTENcomponents, as depicted in . Exemplary sequences are provided in Table 51. In this case, therelease site cleavage ce is incorporated into the CFXTEN that contains an amino acid sequencethat is recognized and cleaved by the FXIa protease (EC 3.4.21.27, Uniprot P03951). Specifically theamino acid sequence KLTRAET (SEQ ID NO: 1688) is out after the arginine of the sequence by FXIaprotease. FXI is the procoagulant protease located immediately before FVIII in the intrinsic or tactivated coagulation y. Active FXIa is ed from FXI by proteolytic cleavage of thezymogen by FXIIa. Production of FXIa is tightly controlled and only occurs when coagulation isary for proper hemostasis. Therefore, by incorporation of the KLTRAET cleavage sequence (SEQID NO: 1688), the XTEN domain is only be removed from FVIII rent with activation of theintrinsic coagulation pathway and when coagulation is required physiologically. This creates a situationwhere the CFXTEN fusion protein is processed in one onal manner during the activation of theintrinsic pathway. inal XTEN releasable by FIIa [thrombin]] A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII isd with an XTEN release site cleavage sequence placed in between the FVIII and XTENents, as depicted in . In this case, the release site contains an amino acid sequence that isrecognized and cleaved by the FIIa protease (EC 3.4.21.5, Uniprot P00734). Specifically the sequenceLTPRSLLV (SEQ ID NO: 1618) [Rawlings N.D., et a1. (2008) Nucleic Acids Res, 36: D320], is out afterthe arginine at position 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in thepresence of phospholipids and m and is down stream from factor IX in the coagulation pathway.
Once activated its l role in coagulation is to cleave fibrinogen (, which then in turn, beginsclot ion. FIIa activity is tightly controlled and only occurs when coagulation is ary forproper hemostasis. Therefore, by incorporation of the LTPRSLLV sequence (SEQ ID NO: 1618), theXTEN domain is only removed from FVIII rent with activation of either the extrinsic or intrinsiccoagulation pathways, and when coagulation is required physiologically. This creates a situation whereCFXTEN fusion is processed in one additional manner during the activation of coagulation.
C-terminal XTEN releasable by Elastase-2 A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII iscreated with an XTEN release site cleavage sequence placed in between the FVIII and XTENcomponents, as ed in . Exemplary sequences are provided in Table 51. In this case, therelease site contains an amino acid sequence that is recognized and cleaved by the e1astase-2 protease(EC 3.4.21.37, t P08246). Specifically the sequence LGPVSGVP (SEQ ID NO: 1689) [RawlingsN.D., et a1. (2008) Nucleic Acids Res, 36: D320], is out after position 4 in the sequence. Elastase isconstitutively expressed by neutrophils and is present at all times in the circulation. Its activity is tightlyWO 22617 2012/046326controlled by serpins and is therefore minimally active most of the time. Therefore as the long livedCFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used ination. In a desirable feature of the inventive composition, this creates a circulating pro-drug depotthat constantly es a prophylactic amount of FVIII.
] C-terminal XTEN releasable by MMP-12 A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII iscreated with an XTEN release site cleavage sequence placed in n the FVIII and XTENcomponents, as depicted in . Exemplary sequences are ed in Table 51. In this case, therelease site contains an amino acid sequence that is recognized and cleaved by the MMP-12 protease (EC3.4.24.65, Uniprot P39900). Specifically the sequence GPAGLGGA (SEQ ID NO: 1690) [RawlingsN.D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 of the sequence. MMP-12 isconstitutively expressed in whole blood. Therefore as the long lived CFXTEN ates, a fraction of itis cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of theinventive composition, this creates a circulating pro-drug depot that constantly releases a prophylacticamount of FVIII.
C-terminal XTEN able by MMP-13 A CFXTEN fusion n ting of an XTEN protein fused to the inus of FVIII iscreated with an XTEN release site cleavage sequence placed in between the FVIII and XTENcomponents, as ed in . Exemplary sequences are provided in Table 51. In this case, therelease site contains an amino acid sequence that is recognized and cleaved by the MMP-13 protease (EC3424-, Uniprot P45452). ically the sequence GPAGLRGA (SEQ ID NO: 1691) [Rawlings N.D.,et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4. MMP-13 is constitutively expressedin whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating apool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition,this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.
C-terminal XTEN releasable by MMP-17 A CFXTEN fusion n consisting of an XTEN protein fused to the inus of FVIII iscreated with an XTEN release site cleavage sequence placed in between the FVIII and XTENcomponents, as depicted in . Exemplary sequences are provided in Table 51. In this case, therelease site contains an amino acid sequence that is recognized and cleaved by the MMP-2O protease(EC.3.4.24.-, Uniprot Q9ULZ9). Specifically the sequence APLGLRLR (SEQ ID NO: 1692) [RawlingsN.D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 in the sequence. MMP-17 isconstitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of itis cleaved, creating a pool of shorter-lived FVIII to be used in ation. In a desirable feature of theinventive composition, this creates a circulating pro-drug depot that ntly releases a prophylacticamount of FVIII.
C-terminal XTEN releasable by MMP-20 A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII iscreated with an XTEN release site cleavage sequence placed in between the FVIII and XTENcomponents, as depicted in . ary sequences are provided in Table 51. In this case, therelease site contains an amino acid sequence that is recognized and cleaved by the MMP-2O se(EC.3.4.24.-, Uniprot 060882). Specifically the ce PALPLVAQ (SEQ ID NO: 1693) [RawlingsN.D., et a1. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 (depicted by the arrow). MMP-is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fractionof it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature ofthe inventive composition, this creates a circulating pro-drug depot that constantly es a prophylacticamount of FVIII.
Optimization of the release rate ofXTEN Variants of the foregoing Examples can be created in which the release rate ofXTENincorporated at the C-terminus, the N—terminus, or internal XTEN is altered. As the rate ofXTENrelease by an XTEN release protease is dependent on the sequence of the XTEN release site, by varyingthe amino acid sequence in the XTEN release site one can control the rate ofXTEN release. Thesequence specificity of many proteases is well known in the art, and is documented in l data bases.
In this case, the amino acid specificity of proteases is mapped using combinatorial libraries of substrates[Harris, J. L., et a1. (2000) Proc Natl Acad Sci U S A, 97: 7754] or by ing the cleavage of substratemixtures as illustrated in [Schellenberger, V., et a1. (1993) Biochemistry, 32: 4344]. An alternative is theidentification of optimal protease cleavage sequences by phage display [Matthews, D., et a1. (1993)Science, 260: 1113]. Constructs are made with t sequences and assayed for XTEN release usingstandard assays for detection of the XTEN ptides.
Example 39: Human al Trial Designs for Evaluating CFXTEN comprising FVIII Kogenate® FS is recombinant human coagulation factor VIII, intended for inghemostasis in ilia A subjects. Due to its short half— life, Kogenate is dosed intravenously everyother day for prophylaxis and 8 to every 12 h in treatment of bleeds until asis is achieved. It isbelieved that fusion of one or more XTEN to FVIII improves the half-life of the protein, enabling areduced dosing frequency using such CFXTEN-containing fusion protein compositions.
Clinical trials are designed such that the y and advantages of CFXTEN, relative toKogenate or other commercially available FVIII preparations, can be verified in humans. Such studiescomprises three phases. First, a Phase I safety and cokinetics study in adult patients is conductedto determine the maximum tolerated dose and cokinetics and pharmacodynamics in humans(either normal subjects or patients with ilia), as well as to define potential toxicities and adverseevents to be tracked in future studies. The Phase I studies are conducted in which single rising doses ofCFXTEN compositions are administered by the route (e.g., subcutaneous, intramuscular, orintravenously) and biochemical, PK, and clinical parameters are measured at defined intervals. Thispermits the determination of the m effective dose and the maximum tolerated dose andestablishes the threshold and m concentrations in dosage and circulating drug that constitute thetherapeutic window for the respective components, as well as bioavailability when stered by theintramuscular or subcutaneous routes. From this information, the dose and dose schedule that permitsless frequent stration of the CFXTEN compositions, yet retains the pharmacologic response, isobtained. Thereafter, clinical trials are ted in patients with the condition, verifying theeffectiveness of the CFXTEN compositions under the dose conditions, which can be conducted incomparison to a positive control such as Kogenate to establish the ed properties of the CFXTENcompositions.
Phase II and III clinical trials are conducted in patients suffering from any disease in whichfactor VIII may be expected to provide clinical benefit. For example, the CFXTEN is used in clinicaltrials for treatment of indications approved for use of factor VIII; such indications include bleedingepisodes in ilia A, patients with inhibitors to factor VIII, prevention of bleeding in surgicalinterventions or invasive procedures in hemophilia A patients with tors to factor VIII, treatment ofng episodes in patients with congenital factor VIII deficiency, and prevention of bleeding insurgical interventions or invasive procedures in patients with congenital factor VIII deficiency.
CFXTEN may also be indicated for use in additional patient populations. A phase II dosing study isconducted in hemophilia A patients where pharmacodynamic, coagulation, ng and otherphysiologic, PK, safety and clinical parameters and clinical endpoints riate for trials are measuredas a on of the dosing of the fusion proteins compositions, yielding dose-ranging ation ondoses that is appropriate for a subsequent Phase III trial, in addition to collecting safety data d toe events. The PK parameters are correlated to the physiologic, clinical and safety parameter datato establish the eutic window and the therapeutic dose regimen for the CFXTEN composition,permitting the clinician to establish the appropriate dose ranges for the composition. In one trial,hemophilia A patients with factor VIII inhibitors would be evaluated to ish doses and dose regimenof CFXTEN pharmaceutical compositions that result in achieVing and maintaining hemostasis andpreventing or attenuating bleeding episodes. Finally, a phase III efficacy study is conducted whereinpatients are administered the CFXTEN pharmaceutical composition and a positive control (such as acommercially-available Kogenate) are stered using a dosing schedule deemed appropriate giventhe pharmacokinetic and pharmacodynamic properties of the respective compositions derived from thePhase II findings, with all agents administered for an appropriately extended period of time to e thestudy endpoints. Parameters that are monitored include aPTT assay, one- or age clotting assays,control of bleeding episodes, or the ence of spontaneous bleeding episodes; ters that aretracked relative to the placebo or positive control groups. Efficacy outcomes are determined usingstandard statistical methods. Toxicity and adverse event markers are also be followed in this study toverify that the compound is safe when used in the manner described. In another phase III trial,hemophilia A patients with factor VIII inhibitors would be evaluated to establish the effectiveness ofCFXTEN pharmaceutical compositions in achieVing and maintaining hemostasis and preventing orattenuating bleeding episodes.
Example 40: Analytical size exclusion chromatography ofXTEN fusion proteins withdiverse ds] Size ion chromatography es were performed on fusion proteins containing varioustherapeutic proteins and unstructured inant proteins of increasing length. An exemplary assayused a TSKGel-G4000 SWXL (7.8mm X 300m) column in which 40 ug of purified glucagon fusionprotein at a concentration of 1 mg/ml was separated at a flow rate of 0.6 ml/min in 20 mM phosphate pH6.8, 114 mM NaCl. Chromatogram profiles were monitored using OD214nm and OD280nm. Columncalibration for all assays were performed using a size exclusion calibration standard from BioRad; themarkers include thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44kDa), equine myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographicprofiles of on-Y288, Glucagon-Y144, Glucagon-Y72, Glucagon-Y36 are shown as an overlay in. The data show that the apparent molecular weight of each compound is tional to thelength of the attached XTEN sequence. r, the data also show that the apparent molecular weightof each construct is significantly larger than that expected for a globular protein (as shown bycomparison to the standard proteins run in the same assay). Based on the SEC analyses for all constructsevaluated, including a CFXTEN ition, the apparent molecular weights, the apparent molecularweight factor (expressed as the ratio of apparent molecular weight to the calculated molecular weight)and the hydrodynamic radius (RH in nm) are shown in Table 35. The results indicate that orationof different XTENs of 576 amino acids or greater confers an apparent molecular weight for the fusionprotein of approximately 339 kDa to 760, and that XTEN of 864 amino acids or greater confers anapparent molecular weight greater than approximately 800 kDA. The results of tional increases inapparent molecular weight to actual molecular weight were consistent for fusion proteins d withXTEN from several different motif families; i.e., AD, AE, AF, AG, and AM, with increases of at leastfour-fold and ratios as high as about 17-fold. Additionally, the incorporation ofXTEN fusion partnerswith 576 amino acids or more into fusion proteins with the s payloads (and 288 residues in the caseof glucagon fused to Y288) resulted with a hydrodynamic radius of 7 nm or greater, well beyond theglomerular pore size of approximately 3-5 nm. Accordingly, it is expected that fusion proteinscomprising growth and XTEN have reduced renal clearance, contributing to increased terminal half-lifeand improving the therapeutic or biologic effect relative to a corresponding un—fused biologic payloadprotein.
Table 35: SEC analysis of s ptidesApparentConstruct KEEN or ntTherapeutic Molecular1181011 . MWName PI‘OtelIl Weightpartner (kDa)Factoruct XTEN or Therapeutic Actual Apparent Sgibiliflgi‘ RHmm“ MWName Protein MW (kDa) Weight (11m)partner (kDa)FactorAC89 AF120 Glucagon 14.1 76.4 5.4 4.3AC88 AF108 Glucagon 13.1 61.2 4.7 3.9AC73 AF144 Glucagon 16.3 95.2 5.8 4.7AC53 AG576 GFP 74.9 339 4.5 7.0AC39 AD576 GFP 76.4 546 7.1 7.7AC41 AE576 GFP 80.4 760 9.5 8.3AC52 AF576 GFP 78.3 526 6.7 7.6AC398 AE288 FVII 76.3 650 8.5 8.2AC404 AE864 FVII 129 1900 14.7 10.1AC85 AE864 Exendin-4 83.6 938 11.2 8.9AC114 AM875 Exendin-4 82.4 1344 16.3 9.4AC143 AM875 hGH 100.6 846 8.4 8.7AC227 AM875 IL-1ra 95.4 1103 11.6 9.2AC228 AM1318 IL-1ra 134.8 2286 17.0 10.5 Example 41: Pharmacokinetics of extended polypeptides fused to GFP in cynomolgusmonkeys The pharmacokinetics of GFP-L288, 76, GFP-XTEN_AF576, EN_Y576 andXTEN_AD836-GFP were tested in cynomolgus monkeys to determine the effect of composition andlength of the unstructured polypeptides on PK parameters. Blood samples were analyzed at varioustimes after injection and the tration of GFP in plasma was measured by ELISA using a polyclonaldy against GFP for capture and a biotinylated preparation of the same polyclonal antibody fordetection. Results are summarized in . They show a surprising increase of half-life withincreasing length of the XTEN sequence. For example, a half-life of 10 h was determined for GFP-XTEN_L288 (with 288 amino acid residues in the XTEN). Doubling the length of the unstructuredpolypeptide fusion partner to 576 amino acids increased the half-life to 20-22 h for multiple fusionprotein constructs; i.e., GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTEN_Y576. A further seof the unstructured polypeptide fusion partner length to 836 residues resulted in a half-life of 72-75 h forXTEN_AD836-GFP. Thus, increasing the polymer length by 288 residues from 288 to 576 residuessed in Vivo half-life by about 10 h. However, sing the polypeptide length by 260 residuesfrom 576 residues to 836 residues sed half-life by more than 50 h. These results show that there isa surprising threshold of unstructured ptide length that results in a greater than proportional gain inin vivo half-life. Thus, fusion proteins sing extended, unstructured polypeptides are expected tohave the property of enhanced pharmacokinetics compared to polypeptides of shorter lengths.
Example 42: Serum ity ofXTEN A fusion protein containing XTEN_AE864 fused to the N—terminus of GFP was incubated inmonkey plasma and rat kidney lysate for up to 7 days at 37°C. Samples were withdrawn at time 0, Day 1and Day 7 and analyzed by SDS PAGE followed by detection using n analysis and detection withantibodies against GFP as shown in . The sequence of XTEN_AE864 showed negligible signs ofdegradation over 7 days in plasma. However, XTEN_AE864 was rapidly degraded in rat kidney lysateover 3 days. The in Vivo stability of the fusion protein was tested in plasma s wherein theGFP_AE864 was immunoprecipitated and analyzed by SDS PAGE as described above. Samples thatwere withdrawn up to 7 days after ion showed very few signs of degradation. The resultsdemonstrate the resistance of CFXTEN to degradation due to serum proteases; a factor in theenhancement of pharmacokinetic properties of the CFXTEN fusion proteins. e 43: Increasing solubility and stability of a e payload by linking to XTEN] In order to evaluate the ability ofXTEN to enhance the physicochemical properties ofsolubility and stability, fusion proteins of glucagon plus shorter-length XTEN were prepared andevaluated. The test articles were prepared in Tris-buffered saline at neutral pH and characterization ofthe Gog-XTEN solution was by e-phase HPLC and size exclusion chromatography to affirm thatthe protein was homogeneous and non-aggregated in solution. The data are ted in Table 36. Forative purposes, the solubility limit of unmodified glucagon in the same buffer was ed at 60uM (0.2 mg/mL), and the result demonstrate that for all lengths ofXTEN added, a substantial se insolubility was attained. antly, in most cases the glucagon-XTEN fusion proteins were prepared toachieve target concentrations and were not evaluated to determine the maximum solubility limits for thegiven construct. However, in the case of glucagon linked to the AF-144 XTEN, the limit of solubilitywas determined, with the result that a 60-fold increase in lity was achieved, compared to glucagonnot linked to XTEN. In addition, the glucagon-AF 144 CFXTEN was evaluated for stability, and wasfound to be stable in liquid formulation for at least 6 months under refrigerated conditions and forapproximately one month at 37°C (data not shown).
The data support the conclusion that the linking of short-length XTEN polypeptides to abiologically active protein such as glucagon can markedly enhance the solubility properties of the proteinby the resulting fusion protein, as well as confer ity at the higher protein concentrations.
Table 36: Solubility of Glucagon-XTEN constructsTest Article SolubilityGlucagon 6O uMGlucagon-Y36 >370 [1V1Glucagon-Y72 >293 uVIGlucagon-AF108 >145 uVIGlucagon-AF12O >160 uVIGlucagon-Y144 >497 uVIGlucagon-AE144 >467 uVIGlucagon-AF144 >3600 [1MGlucagon-Y288 >163 uVI Example 44: Analysis of sequences for ary structure by prediction algorithms Amino acid ces can be assessed for ary structure via certain computer programsor algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry,13: ) and the Garnier-Osguthorpe-Robson, or “GOR” method (Garnier J, Gibrat JF, Robson B.(1996). GOR method for predicting protein secondary structure from amino acid sequence. Methodsl 0-553). For a given sequence, the algorithms can predict whether there exists some orno secondary structure at all, expressed as total and/or percentage of residues of the sequence that form,for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to resultin random coil formation.
Several representative sequences from XTEN “families” have been assessed using twoalgorithm tools for the Chou-Fasman and GOR methods to assess the degree of secondary ure inthese sequences. The Chou-Fasman tool was provided by William R. Pearson and the University ofVirginia, at the pport” et site, URL located on the World Wide Web at.fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it existed on June 19, 2009. TheGOR tool was provided by Pole lnformatique Lyonnais at the Network Protein Sequence Analysisinternet site, URL located on the World Wide Web at .npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.p1 as itexisted on June 19, 2008.
As a first step in the analyses, a single XTEN sequence was analyzed by the two algorithms.
The AE864 composition is an XTEN with 864 amino acid residues created from multiple copies of four12 amino acid sequence motifs consisting of the amino acids G, S, T, E, P, and A. The sequence motifsare characterized by the fact that there is limited repetitiveness within the motifs and within the overallsequence in that the sequence of any two consecutive amino acids is not ed more than twice in anyone 12 amino acid motif, and that no three uous amino acids of full-length the XTEN are identical.
Successively longer portions of the AP 864 sequence from the N—terminus were analyzed by the Chou-Fasman and GOR algorithms (the latter es a minimum length of 17 amino acids). The sequenceswere analyzed by entering the FASTA format sequences into the prediction tools and running theanalysis. The results from the analyses are presented in Table 37.
The results indicate that, by the Chou-Fasman calculations, short XTEN of the AE and AGfamilies, up to at least 288 amino acid residues, have no alpha-helices or beta-sheets, but s ofpredicted percentage of random coil by the GOR thm vary from 78-99%. With sing XTENlengths of 504 residues to r than 1300, the XTEN analyzed by the Chou-Fasman algorithm hadted percentages of alpha-helices or beta-sheets of 0 to about 2%, while the calculated percentagesof random coil increased to from 94-99%. Those XTEN with alpha-helices or beta-sheets were thosesequences with one or more instances of three contiguous serine residues, which resulted in predictedbeta-sheet formation. However, even these sequences still had approximately 99% random coilformation.
The data provided herein suggests that 1) XTEN created from le sequence motifs of G,S, T, E, P, and A that have limited repetitiveness as to contiguous amino acids are predicted to have verylow amounts of alpha-helices and beta-sheets; 2) that increasing the length of the XTEN does notappreciably increase the probability of helix or beta-sheet formation; and 3) that progressivelyincreasing the length of the XTEN sequence by addition of non-repetitive s consisting of theamino acids G, S, T, E, P, and A results in increased tage of random coil formation. Resultsfurther indicate that XTEN sequences defined herein (including e. g., XTEN created from cemotifs of G, S, T, E, P, and A) have limited repetitiveness (including those With no more than twoidentical contiguous amino acids in any one motif) are expected to have very limited secondary structure.
Any order or combination of sequence motifs from Table 3 can be used to create an XTEN polypeptidethat will result in an XTEN sequence that is substantially devoid of secondary structure, though threecontiguous serines are not preferred. The unfavorable property of three contiguous series however, canbe ameliorated by sing the length of the XTEN. Such sequences are expected to have thecharacteristics described in the CFXTEN embodiments of the invention disclosed herein.
Table 37: CHOU-FASMAN and GOR prediction calculations of polypeptide seguencesSEQ ID No. Chou-Fasman GORSEQ NAMEN0: Residues Calculation CalculationAE36-- R'd651 ue ttl:H:0E:00 a S1489 36 94.44%LCW0402_002 percent: H: 0.0 E: 0.0AE36-. R'd651 ue ttl:H:0E:00 a S1490 36 94.44%LCW0402_003 t: H: 0.0 E: 0.0AG36-- R'd“1 ue 0E:00 a S1491 36 77.78%LCW0404_001 percent: H: 0.0 E: 0.0AG36-. Residue totals: H: 0 E: 01492 36 83.33 %LCW0404_003 percent: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AE42_1 1493 42 90.48%percent: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AE42 1 1494 42 90.48%— percent: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AG42 1 1495 42 88.10%— t: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AG42_2 1496 42 8810‘Vi 0percent: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AE144 1497 144 98.61%t: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AG144 1 1498 144 91.67%_ percent: H: 0.0 E: 0.0Residue totals: H: 0 E: 0AE288 1499 288 99.31%percent: H: 0.0 E: 0.0Residue : H: 0 E: 0AG288_2 1500 288 92 71Ipercent: H: 0.0 E: 0.0Residue : H: 0 E: 0AF504 1501 504 94.44%percent: H: 0.0 E: 0.0Residue totals: H: 7 E: 0AD 576 1502 576 99.65%)0percent: H: 1.2 E: 00AE576 1503 576 Residue totals: H: 2 E: 0 99.65%WO 22617 SEQ I No. Ch0u~Fasman GORSEQ NAMEN0: Residues Calculation Calculationt: H: 0.4 E: 0.0Res'd1 ue Gastt1:H: 0 E: 3AG576 1504 576 99.31%percent: H: 0.4 E: 0.5Residue : H: 2 E: 0AF540 1505 540 99.65percent: H: 0.4 E: 0.0Res1due totals: H: 0 E: 0AD836 1506 836 98.44%percent: H: 0.0 E: 0.0R'd651 ue tt1:H:2E:30 a SAE864 1507 864 99.77%t: H: 0.2 E: 0.4Residue totals: H: 2 E: 0AF864 1508 875 95.20%)0percent: H: 0.2 E: 00R'd651 ue tt1:H:0E:00 a SAG864 1509 864 94.91%percent: H: 0.0 E: 0.0R'd651 ue tt1:H:7E:30 a SAM875 1510 875 98.63%percent: H: 0.8 E: 0.3R”due' t0 a St 1 :H: 7 E: 0AM1318 1511 1318 99.17%percent: H: 0.7 E: 0.0Residue totals: H: 4 E: 3AM923 1512 924 98.70%)0percent: H: 0.4 E: 03Residue totals: H: 8 E: 3AE912 1513 913 )0percent: H: 0.9 E: 0.3Residue totals: H: 0 E: 0BC 864 1514 99.77%)0percent: H: 0 E; 0H: alpha-helix E: beta-sheet Example 45: Analysis of polypeptide sequences for repetitiveness In this e, different polypeptides, including several XTEN sequences, were assessed forrepetitiveness in the amino acid sequence. Polypeptide amino acid sequences can be assessed forrepetitiveness by quantifying the number of times a shorter subsequence appears within the overallpolypeptide. For example, a polypeptide of 200 amino acid residues length has a total of 165overlapping 36-amino acid “blocks” (or “3 6-mers”) and 198 3-mer “subsequences”, but the number ofunique 3-mer subsequences will depend on the amount of repetitiveness within the sequence. For theanalyses, different polypeptide sequences were assessed for repetitiveness by determining thesubsequence score obtained by ation of the following equation:@333 >, , . .)Subsequence score = “33"“: {Zalfiffiig lwherein: m = (amino acid length of polypeptide) — (amino acid length of subsequence) +1; and Countl- = cumulative number of occurrences of each unique subsequence withinsequence,-In the es of the t e, the subsequence score for the polypeptides of Table 38 weredetermined using the foregoing equation in a er program using the algorithm depicted in ,n the subsequence length was set at 3 amino acids. The resulting subsequence score is a reflectionof the degree of repetitiveness within the polypeptide.
The results, shown in Table 38, indicate that the unstructured polypeptides ting of 2 or 3amino acid types have high subsequence scores, while those of consisting of the 12 amino acid motifs ofthe six amino acids G, S, T, E, P, and A with a low degree of internal repetitiveness, have subsequencescores of less than 10, and in some cases, less than 5. For example, the L288 sequence has two aminoacid types and has short, highly repetitive sequences, resulting in a subsequence score of 50.0. Thepolypeptide J288 has three amino acid types but also has short, repetitive sequences, resulting in asubsequence score of 33.3. Y576 also has three amino acid types, but is not made of al repeats,reflected in the subsequence score of 15.7 over the first 200 amino acids. W576 consists of four types ofamino acids, but has a higher degree of internal repetitiveness, e. g., “GGSG” (SEQ ID NO: 1694),”,ing in a subsequence score of 23.4. The AD576 consists of four types of 12 amino acid motifs,each consisting of four types of amino acids. Because of the low degree of internal repetitiveness of theindividual motifs, the overall subsequence score over the first 200 amino acids is 13.6. In contrast,XTEN’s consisting of four motifs contains six types of amino acids, each with a low degree of internalrepetitiveness have lower subsequence scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5), whileXTEN consisting of four motifs containing five types of amino acids were ediate; i.e., AE864,with a score of 7.2.
Conclusions: The results indicate that the combination of 12 amino acid subsequence ,each consisting of four to six amino acid types that are non-repetitive, into a longer XTEN polypeptideresults in an overall sequence that is substantially non-repetitive, as ted by overall subsequencescores less than 10 and, in many cases, less than 5. This is despite the fact that each subsequence motifmay be used multiple times across the sequence. In st, polymers created from smaller numbers ofamino acid types resulted in higher subsequence , with polypeptides consisting of two amino acidtype having higher scores that those consisting of three amino acid types.
Table 38: Subseguence score calculations of ptide seguencesSeq Name SEQ ID NO: ScoreJ288 1515 33.3K288 1516 46.9L288 1517 50.0Y288 1518 26.8Q576 1519 18.5U576 1520 18.1W576 1521 23.4Y576 1522 15.7AE288 1523 6.0AG288_1 1524 6.9AD576 1525 13.6AE576 1526 6.1Seq Name SEQ ID NO: ScoreAF54O 1527 8.8AF504 1528 7.0AE864 1529 6.1AF864 1530 7.5AG864 1531 7.2AG868 1532 7.5AM875 1533 4.5AE912 1534 4.5AM923 1535 45AM1296 1536 4.5 Example 46: Calculation of TEPITOPE scores TEPITOPE scores of 9mer peptide sequence can be calculated by adding pocket potentials asdescribed by Sturniolo [Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555]. In the present Example,separate Tepitope scores were calculated for dual HLA alleles. Table 39 shows as an e thepocket potentials for HLA0101 B, which occurs in high frequency in the Caucasian population. Toate the TEPITOPE score of a e with sequence Pl-P2-P3-P4-P5-P6-P7-P8-P9, thecorresponding individual pocket potentials in Table 39 were added. The HLA0101B score of a 9merpeptide with the sequence FDKLPRTSG (SEQ ID NO: 1695) is the sum of 0, -l.3, 0, 0.9, 0, -l.8, 0.09, 0, To evaluate the TEPITOPE scores for long es one can repeat the process for all 9mersubsequences of the sequences. This s can be repeated for the proteins encoded by other HLAalleles. Tables 40-43 give pocket potentials for the protein ts of HLA alleles that occur with highfrequency in the Caucasian population.
TEPITOPE scores calculated by this method range from approximately -10 to +10. r,9mer peptides that lack a hydrophobic amino acid (FKLMVWY (SEQ ID NO: 1696)) in P1 positionhave calculated TEPITOPE scores in the range of -1009 to -989. This value is biologically meaninglessand reflects the fact that a hydrophobic amino acid serves as an anchor residue for HLA binding andpeptides lacking a hydrophobic residue in P1 are considered non binders to HLA. Because most XTENsequences lack hobic residues, all combinations of 9mer subsequences will have TEPITOPEs inthe range in the range of -1009 to -989. This method confirms that XTEN polypeptides may have few orno predicted T-cell epitopes.
Table 39: Pocket otential for HLA0101B allele.
WEE”—mnnninnI-I—mnnnlnnl-IWO 22617 Amino Acid P1 P2 P3 P4 P5 P6 P7 P8 P9D _ -2 _Table 40: Pocket potential for HLA0301B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9A -999 O O O - O O - OC -999 O O O - O O - O.3 -Amino acidAmino acidEQTJD'J 9.0.0.0 OOLAOOI—I‘ .0ch NNOO‘N‘ ._I,'_.<'3._I H'oxkom -I>UIt—t-I> o I0 llll llll I—IN HN' llll ' III—I I—kI—I- \II—I I ._I 0 00 I I _o U.)W I—II—I I—II—I oI—ILI] I I—k \l I IIN9 AI—I IO U) I Io U)gr II I—KI—K I—I .I—K )—I )—I .I—K .5 9.0\OOO‘ II II I—KI—K 55' $3.0 oo\l' II I OI .._...5O 00 O U} 0 0 )—k.0..00. . U) 0 O\ .'_I .5MPG/Ow )—I N. I I H LII I .00]..s5 U) .0 [\D .0 00 I )—k I .0 [\D I .0 \lIfi O O O \l I I—I 0 IO I—I I .'_I N00;. 5N;_‘I—I oOmll I—AO_ No II ._o_"xo II I no 5...\II )—I .5O 0 .0 00 I I—I O\ I I I—I LI] I I—I [\D I I I-ITable 42: Pocket potential for HLA0701B allele.
Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9A OAmino acid p—I "UN "U L») "d.5 *UU} "U OK "U x] "U 00 "UQl D—I D—I I I L» .5W p—Ip—I p—Ip—I OD—IL11 I H. L») I II I—‘O I—‘LII ON LII-P I I H. I—IEb II I—II—I L»)._I .I—I )—I )—I .I—I.5 II .053. #00 II II .053. 000 I—‘N OON IIO 00 0 LA I ._I ._I I IO O\ D—I .5 I I0 L11MPG/Orv NI—A .NN. ll K I—‘Lll III II _b—KD—K b—KD—K OI—I "\I'_." III II .99.. cowIo L» 00Mac D—I LII o O\ o -I> <5 L»,4 o o D—I -I> I ('3 I—A o O I 0 -'>.N D—I .0 m .0. \o I .0_. 5. o I N.0L <5 I—A O I b—k I_I I IO 0 I—I -I> I O 000 0 O 00 IO Lo I | ._I I—I \l I I—‘ l—‘Table 43: Pocket ial for HLA1501B allele.
Amino acid Example 46: Assessment of insertion ofXTEN into permissive loops.
XTEN AE42-4 Insertion The construction and expression of FVIH with XTEN AE42 insertions were described inExample 17 and 24. Thus, Where residue X designates the site of insertion and residue Z designates thenext residue in the native FVIII ptide sequence, the polypeptide resulting from insertion ofXTENAE42 would contain the sequence:X-GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS-Z (SEQ ID NO: 1697) 16 different sites in the FVIII sequence were selected for XTEN AE42 insertion, and thesewere designed Batch 1. An additional 21 sites selected for XTEN AE42 insertion were designed Batch 2.tively, the Batch 1 and Batch 2 sites represent 12 sites in the Al domain, 7 sites in the A2 domain,sites in the A3 domain, 4 sites in the Cl domain, and 3 sites in the C2 . Locations of Batch 1and 2 sites in the 3-D structure of FVIII are depicted in .
The location of these Batch 1 and Batch 2 insertion sites results in 37 constructs designatedpSDOOOl- 4, pSDOOO9- pSDOOl2, pSDOO23- pSDOO32, pSDOO34- pSDOO63 [the ingranges include all intermediate numbers, as well], the sequences of which are set forth in Table 21 andthe insertions sites of which are set forth in Table 23.
In Vitro assays To assess FVIII tolerability to XTEN AE42-4 insertion, the FVIII actiVity in e mediasamples from FVIII-XTEN cell cultures was analyzed using a FVIII chromogenic assay. Antigenexpression levels were analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain)ELISA.
FVIII ActiVity Measurement by genic Assay] The FVIII actiVity was measured using the COATEST® SP FVIII kit from DiaPharma (lot#N0890l9) and all incubations were performed on a 37°C plate heater with shaking. Cell culture harvestsfrom transient transfection media of FVIII-XTEN AE42-4 ts from 6 well plates were diluted to thedesired FVIII actiVity range using lx FVIII T® buffer. FVIII standards were prepared in 1xFVIII COATEST® buffer containing mock transfection media with matching culture mediaconcentration as the testing sample. The range of recombinant Factor VIII I) standard was from100 mIU/mL to 0.78 mIU/mL. The standards, diluted cell culture samples, and a pooled normal humanplasma assay control were added to Immulon® 2HB 96-well plates in duplicates (25 uL/well).
] Freshly prepared IXa/FX/Phospholipid mix (50 uL), 25 [LL of 25mM CaCl2, and 50 [LL of FXasubstrate were added sequentially into each well, with 5 minutes incubation between each addition. Afterincubating with the substrate, 25 [LL of 20% acetic acid was added to terminate the color reaction, and theabsorbance at 405 nm was measured with a SpectraMAX® plus (Molecular DeVices) instrument.
Data analysis was performed using SoftMax Pro software on 5.2). The Lowest Level ofQuantification (LLOQ) was 39 mIU/mL. s are presented in Table 22.
Expression Measurement by FVIII-HC and FVIII-LC ELISA Expression of variants was quantified using ELISA. The FVIII antigen expression levels ofDNA constructs corresponding to XTEN ions in the Al and A2 domains of FVIII were analyzed byFVIII-LC ELISA. The FVIII antigen expression levels of DNA constructs corresponding to XTENinsertions in the A3, C1 and C2 s of FVIII were analyzed by FVIII-HC ELISA. Results arepresented in Table 22.
WO 22617 FVIII-XTEN antigens in cell culture media after harvest were captured by GMA011 antibodies(Green Mountain Antibodies) for FVIII-LC ELISA) or by GMA016 antibodies (Green MountainAntibodies) for FVIII-HC ELISA. Immulon® 2HB 96-well plates were coated with 100ul/well of anti-FVIII antibody (2ug/ml) by overnight incubation at 40C. Plates were then washed four times withPhosphate Buffer saline with Tween-20 (PBST) and blocked with blocking buffer (PBST with 10% heatinactivated horse serum) for 1 hour at room temperature.
Cell culture harvests from transient ection media of FVIII-XTEN variants from a 6-wellplate were diluted to the desired FVIII antigen range using 1x blocking buffer. FVIII standards wereprepared in 1x FVIII blocking buffer containing mock ection media with matching mediaconcentration as the g samples. The range of rFVIII standard was from 50 ng/mL to 0.39 ng/mL.
Standards, diluted cell culture samples, and a pooled normal human plasma assay control wereadded into Immulon® 2HB 96-well plates in duplicates (100 uL/well) and incubated at 370C for 2 hours.
Following four times g with PBST, 100 pl of HRP-sheep FVIII antibody (AffinityBiologicals, F8C-EIC-D) were added into each well and plates were incubated for 1 hour at 370C. Afteranother four washes with PBST, 100 pl ofTMB Super Sensitive Substrate ) were added to eachwell, followed by 5-10 min color development. To ate the color on, 50 [LL of H2SO4 wereadded to each well, and the absorbance of at 450 nm was measured with a SpectraMAX plus (MolecularDevices) instrument.
Data analysis was performed using SoftMax Pro software (version 5.4). The Lowest Level ofQuantification (LLOQ) was 0.0039 ug/mL. Results are presented in Table 22.
Permissive sites into which XTEN sequences were inserted without eliminating procoagulantactiVity of the recombinant protein, or the y of the recombinant proteins to be expressed in the hostcell were clustered within loops in each of the three A domains of FVIII. shows the location ofinsertion sites in the recombinant FVIII proteins that showed FVIII ty on domains A1, A2 and A3. shows a structural representation depicting the location of insertion sites in the recombinantFVIII proteins that showed FVIII actiVity.
The sive sites clustered in t exposed, highly flexible surface loops (XTENpermissive loops). The A1 domain loops were located in a region corresponding imately to aminoacid positions 15 to 45, and 201 to 232, respectively, in the sequence of mature human FVIII ().
The A2 domain loops were d in a region corresponding imately to amino acid positions 395to 421, and 577 to 635, respectively, in the sequence of mature human FVIII (). The A3 domainloops were located in a region corresponding approximately to amino acid positions 1705 to 1732, and1884 to 1917, respectively, in the sequence of mature human FVIII (). FIGS. 37A and 37B showthe location of the XTEN permissive loops relative to secondary structure elements in the tridimensionalstructure of FVIII.
Example 47: CFXTEN with insertions ofXTEN having 144 amino acids Analysis of the preliminary data presented above (Example 46) suggested the existence ofdefined regions within the linear ptide sequences and 3-D structures of the FVIII A domains thatcan accommodate the insertion ofXTEN sequences. To test this hypothesis and further define theboundaries of putative regions that can accommodate the insertion ofXTEN sequences without loss ofFVIII activity, 23 additional insertion sites not present in either Batch 1 or 2 were chosen and designatedBatch 3.
Batch 3 constructs were generated by the insertion of a 144 residue XTEN AE polypeptide,comprising amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), Thr (T), and Glu (E), or a 144 residueXTEN AG polypeptide, comprising amino acid es Gly (G), Ala (A), Pro (P), Ser (S), and Thr (T).
Five different version of the 144 residue AE ptide were generated and designated XTEN-AEl44-2A, XTEN-AEl44-3B, XTEN-AEl44-4A, XTEN-AEl44-5A, XTEN-AEl44-6B. The amino acidces are as set forth in Table 4. Five different versions of the 144 residue polypeptide weregenerated and designated XTEN-AGl44-l, XTEN-AGl44-A, XTEN-AGl44-B, XTEN-AGl44-C, andXTEN-AGl44-F. The amino acid sequences are as set forth in Table 4.
] The 144 residue XTEN encoding DNA sequence was introduced by the chemical synthesis ofDNA segments (DNA 2.0, d City, CA) spanning the t unique ction sites within thebase vector on either side of the site of insertion.
The DNA sequences corresponding to the XTEN 144 peptides were inserted such that theresulting DNA construct would encode a FVIII protein in which the XTEN 144 protein sequence isinserted immediatelym the residue indicated in the site selection, and flanked by AscI and X1101 sites.
In on to these sites, those sites from Batch 1 and 2 at which insertion of the XTEN AE42polypeptide did not h FVIII procoagulant acitivity were modified by excision of the AE42polypeptide encoding DNA t with restriction enzymes AscI and X7101, and introduction ofXTENAEl44 and XTEN AGl44 coding ces at the same sites. The location of these Batch 1, Batch 2 andBatch insertion sites is summarized in Table III. presents a structural representation of FVIIIshowing the on of the XTEN l44 insertion sites.
A total of 48 constructs with 144 XTEN inserts were created. The constructs are pSDOOOl-pSDOOO4, pSDOOO9-pSDOOl2, pSDOO23-63 [the foregoing ranges include all intermediate numbers, aswell], the sequences of which are set forth in Table 21 and the insertion sites of which are detailed inTable 22.
Expression of FVIII-XTEN 144 Variants FVIII variants with XTEN l44 insertions were tranfected into HEK293F cells (Invitrogen,ad, CA) using hyleneimine (PEI, Polysciences Inc. Warrington, PA) or Lipofectaminetransfection reagent (Invitrogen, Carlsbad, CA). The transiently transfected cells were grown in 293 FreeStyle medium or a e of 293 Free Style and CD Opti CHO media (Invitrogen, Carlsbad, CA). Thecell culture medium was harvested 3-5 days after transfection and analyzed for FVIII expression bychromogenic FVIII activity assay and FVIII ELISA conducted as bed herein.
Cell culture media from transient transfection were concentrated 10-fold in Centricon® spincolumns (lOOkd cut-off). Concentrated material was then flash frozen and stored at -80°C for future invitro analysis and in vivo PK studies.
In vitro assays To assess FVIII tolerability to insertions, the FVIII activity in culture media samples from cellcultures was analyzed using a FVIII chromogenic assay. Antigen expression levels were analyzed byFVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.
FVIII Activity Measurement by Chromogenic Assay and Expression Measurement by FVIII-HC and FVIII-LC ELISA Chromogenic and ELISA assay methods were conducted as described. The s obtained aresummarized in Table 23. sive sites into which XTEN sequences were inserted without ating procoagulantactivity of the recombinant protein, or the ability of the inant proteins to be expressed in the hostcell clustered within loops in each of the three A domains of FVIII. The same XTEN permissive loopregions tolerating the shorter XTEN sequences inserted were found to tolerate the insertion of the longerXTEN sequences. shows the location ofXTEN 144 insertion sites in the recombinant FVIIIproteins that showed FVIII activity on domains A], A2 and A3. shows a structuralentation depicting the location of insertion sites in the recombinant FVIII ns that showedFVIII activity.
These observation indicate that two regions within each of the A domains of FVIII are able toaccommodate insertion ofXTEN sequences without loss of FVIII or activity. A structuraldepiction of these so-called XTEN permissive loops (FIGS. 40 and 4]) demonstrate that they occupystructurally analogous ons in each of the A domains and project from one face of the FVIIImolecule. The identified XTEN permissive loops correspond to highly flexible loops located betweenbeta strands in the three-dimensional structures of the Al, A2, and A3 domains, as shown in FIGS. 37Aand 37B.
The in vivo evaluation ofXTEN 144 insertions on FVIII ife Extension, as determined bypharmacokinetics, is described in Example 32.
Example 48: Rescue or enhancement of FVIII expression by insertion of an XTENsequence within the a3 acidic peptide region of FVIII. nt HEK293 cells were transfected (as described in e 24) with FVIII-XTEN DNAconstructs in which the coding ce of a B-domain deleted factor VIII contained 2 to 4 XTENinsertions of 144 amino acid residues each, of composition and insertion location as indicated in Table44, below. At 5 days post-transfection, cell culture supernatants were assayed for FVIII activity by thechromogenic assay (as described in Example 25). Results are shown in Table 44.
Table 44. Expression levels of FVIII Activity by CFXTEN variants containing an XTEN at position1720 and one, two, or three additional XTEN insertions.
Construct , on, and Type ofXTEN InsertionActivityName (mm/mm1720LSD0040.002 26 AGl44 AGl44 175_—403 AE144_ 1720 __WO 22617 AG1441656 1720LSD0045.002 AG144 AG144 25981656 1720PSD080.002 26 AG144 AG144 AG144 10811656 1720PSD083.001 403 AE144 AG144 AG144 7891720PSD082.001 26 AG144 AG144 1900 AE144 <LLOQ1656 1720.003 26 AG144 AG144 AG144 1900 AE144 316 For the e of comparison, all FVIII-XTEN constructs had an AG144 XTEN insertion atamino acid position 1720 (numbered relative to full-length factor VIII) within the A3 domain.
Expression levels of FVIII-XTEN varians were determined by chromogenic assay and expressed in unitsof mIU/mL. Constructs with a single additional XTEN insertion at either position 26 in the A1 domain(LSD0040.002) or on 403 in the A2 domain (LSD0041.008) yielded expression levels of 175 and279 mIU/mL, respectively. In contrast, a construct with a single additional XTEN insertion at on1656 within the a3 acidic peptide yielded an expression level of 2598 mIU/mL, demonstratingenhancement of expression level for the a3 XTEN insertion construct relative to the A1 and A2 insertionconstructs. In addition, in comparison to the FVIII-XTEN uct with XTEN insertions at ons 26in the A1 domain and 1720 in the A3 domain 40.002), the construct with an additional XTENinsertion at position 1656 within the a3 acidic peptide region (PSD080.002) yielded significantly higherexpression (175 and 1081 mIU/mL, respectively). Consistent with these findings, the construct withXTEN insertions at positions 403 in the A2 domain and 1720 in the A3 domain (LSD0041.008) yieldedan expression level of 279 mIU/mL, s an additional XTEN insertion at position 1656 within the a3acidic peptide region (PSD083.001) resulted in an increase in the expression level to 789 mIU/mL.
Lastly, the FVIII-XTEN construct with an XTEN ion at position 26 within the A1 domain and twoXTEN ions at positions 1720 and 1900 within the A3 domain (PSD082.001) did not yield activityabove the lower limit of quantitation. However, the FVIII-XTEN construct with an additional XTENinsertion within the a3 acidic peptide region (PSD090.003) resulted in detectable activity, demonstratingthat inclusion of an XTEN sequence within the a3 domain can result in recovery of expression (asmeasured by activity) in FVIII-XTEN constructs that are ise expressed at levels below the lowerlimit of quantitation. Under the conditions of the experiment, the results support the sion thatinsertion ofXTEN at the 1656 position and, by extension, within the a3 region, results in enhancedexpression of procoagulant FVIII-XTEN compositions.
Example 49: Effect ofXTEN insertion 0n FVIII ty measured by aPTT A one stage activated partial prothrombin (aPTT) coagulation assay was employed in additionto the chromogenic assay (as described in Example 25) to determine FVIII ty of various FVIII-XTEN fusion proteins.
Method: The FVIII-XTEN aPTT activity was measured using the Sysmex CA—lSOO instrument(Siemens care Diagnostics Inc.,Tarrytown, NY). To create a standard curve for the assay, WHOfactor VIII standard was diluted with 2% mock transfection media to 100 mU/mL and a two-fold serialdilution series was then performed, with the last standard being 0.78 mU/mL. FVIII-XTEN cell culturesamples were first diluted at 1:50 with aPTT assay buffer, further dilutions were made with 2% mocktransfection media when needed.
After dilution, the aPTT assay was performed using Sysmex instrument as follow: 50 [ll of dilutedstandards and samples were mixed with 50 ul human FVIII def1cient plasma and then 50 ul of aPTTreagent. The mixture was incubated at 37°C for 4 min, and following tion, 50 ul of CaClz wasadded to the mixture, and the clotting time was measured immediately.
To ine test samples FVIII actiVity, the clotting time of the standards were plotted using semi-logscale (Clotting time: ; Standard concentration: Log) to extrapolates the equation between ngtime and FVIII actiVity, and FVIII-XTEN actiVity was then calculated t the standard curve. Theassay sensitiVity was 40 mU/mL factor VIII.
RLults: The results are summarized in FIGS. 44-46. When single XTEN of 144 or 288 aminoacids were inserted into the FVIII, all of the FVIII-XTEN fusion proteins exhibiting ty in thechromogenic assay were also active in aPTT assay. The aPTT actiVity ed the trend ofchromogenic assay, for example, those molecules that showed low FVIII actiVity in the genicassay also had low aPTT values. Generally, the aPTT results for the fusion proteins were lower thanthose obtained by the chromogenic assay, with a chromogenic to aPTT ratio of 1.1 up to 2.2, asillustrated in , for the single XTEN insertions. The FVIII-XTEN fusion proteins with multipleXTEN insertions, in general, showed further reductions in aPTT actiVity in comparison to chromogenicassay. Assays of FVIII-XTEN with two XTEN insertions showed actiVity with all constructs, but withchromogenic/aPTT ratios approaching 4, in some instances (). Assays of XTEN with somethree XTEN insertions also showed ty in both assays, with chromogenic/aPTT ratios approaching 5,in some instances (), while the ratios for the BDD-FVIII control were more comparable (rightside of FIG 46). Additionally, the site ofXTEN insertion appeared to contribute to the differences seenbetween aPTT and chromogenic actiVities. For example, while some les with 2 XTEN insertionsresulted in up to 4-fold lower actiVity than chromogenic values, the aPTT actiVity of other FVIIImolecules with 2 XTEN were fairly comparable to chromogenic activity (). Some moleculeswith 3 XTEN insertions showed up to 5 —fold lower than chromogenic activities, other FVIII moleculeswith 3 XTEN have aPTT actiVity less than 2-fold lower than chromogenic actiVity (). Under theconditions of the experiment, the results t the conclusion that FVIII-XTEN fusion nconstructs do retain procoagulant actiVity, but that the chromogenic assay lly provides higheractiVity levels than that in the aPTT assay system ed in the study.
Example 50: Evaluations of the Effect ofXTEN Insertion Site onf FVIII Half-lifeExtension Methods: Six FVIII-XTEN fusion proteins with single XTEN AG-144 insertions at definedlocations were tested in FVIII/VWF DKO mice (as generally described in Example 32) to te theeffect ofXTEN insertion site on FVIII half-life. Six representative XTEN variants d in table 1) withXTEN insertion in either within A1, A2, a3, A3-region1 (A3-R1), A3-region 2 (A3-R2) or at the C-terminus were selected for this study, and BDD-FVIII generated from the base vector was used as thecontrol. FVIII/VWF DKO mice were treated with a single intravenous administration of transienttransfaction cell e media concentrate from the six FVIII-XTEN constructs (or positive controlmedia) at 100-200 IU/kg, and plasma samples were subsequently ted at 5min, 7 hours and 16 hourspost-dosing. Plasma FVIII activity was tested using the FVIII chromogenic assay and XTEN half-life was estimated using the WinNonlin program. The study data are summarized in Table 45 and FIG RLults: A significantly longer half-life was observed for all FVIII-XTEN variants testedcompared to BDD-FVIII control, but the degree of the half-life increase varied, with the variant withXTEN at the 403 insertion site conferring the least half-life extension at 10-fold (in comparison tocontrol), while the 1900 insertion variant conferred the most half-life extension at 18-fold. Thedifferences ofXTEN ion site on FVIII ife extension may reflect the roles of different FVIIIdomains in FVIII clearance in vivo.
Table 45: FVIII-XTEN single AG-144 insertion variants PK in FVIIINWF DKO miceBDD- pSD pSD- pSD- pSD- pSD- pSD-TreatmentFVIII -050 0003 0039 0010 063 014Insertion site None 26 403 1656 1720 1900 CTRecovery 21.3 33.8 34.8 36.0 33.6 39.6 32.4th/rz 0.25 3.15 2.4 3.3 4.28 4.54 3.91t1/2 Increase13 10 13 17 18 16(fold)] Example 51: Evaluations of the Additive Effect ofXTEN ions 0n FVIII Half-lifeion.
Methods: To evaluate the effects of multiple XTEN insertions on FVIII-XTEN fusion proteinhalf-life, the half-livesof FVIII-XTEN variants with 1-3 XTEN insertions were determined in FVIII-XTEN DKO mice using the cell culture concentrate from five ucts (as generally described inExample 32). Five XTEN variants were tested in the study: pSD-062, with AE144 ion atposition 1900 (numbered relative to full-length factor VIII); 05 with AE144 in the FVIII Bdomain (B-domain amino acid position 745); pSD-0019 with AE288 at the FVIII C-terminus (CT);LSD0003.006 with AE144 inserted in the B-domain and AE288 inserted at the C-terminus, andLSD0055.021 with three XTEN ofAE144, AE144, and AE288 inserted at position 1900, with the Bdomain and at the C-terminus. The FVIII-XTEN half-life values were estimated using the WinNonlinprogram.
Results: The study results are ized in Table 46, and the PK curves are shown in . The study results clearly demonstrated the additive effect of multiple XTEN insertions on FVIII half-life extension. With single XTEN insertions, the half-life of FVIII was ed from 0.25 hr to 3.2-4.0hr, a 13 tol6-fold increase. When the B and CT XTEN insertions were combined together, the FVIIIhalf-life was further extended to 10.6 hr, a 42-fold prolongation. Finally, in the case of a third XTENinsertion added at position 1900 to the B/CT construct, the half-life reached 16 hr in the FVIII-VWFDKO mice, a 64-fold increase.
Table 46: Additive effect ofXTEN insertions 0n FVIII tm in FVIIINWF DKO miceBDD- pSD pSD— pSD— LSD- LSD-FVIII -062 0005 0019 0003.006 0055.021Insertion s1te0.25 3.8 3.2 4.0 10.6 16.0t1/2Increase13(fold) - Example 52: Evaluation of FVIII-XTEN Interference with the Binding of Anti-FVIIIAntibodies using the Bethesday Assay] The ability ofXTEN insertions in the FVIII molecule to interfer with binding by pre-existinganti-FVIII antibodies to the XTEN fusion n was evaluated in order to determine their utilityin treating patients with anti-FVIII inhibitory antibodies.
Methods: To assess the binding of anti-FVIII antibodies, two FVIII-XTEN variants 8,with 144 XTEN inserted at the locations of 26/403/1656/1900; and PSD-090, with 144 XTEN inserted atthe locations of 26/1656/1720/1900) were tested in comparison with Refacto (a marketed rFVIII) againstplasma samples from three hemophilia A patients with factor VIII inhibitors (designated 04-483, 05-505,and -2079), as well as a sheep anti-FVIII poly-clonal antibody from Affinity Biologicals Inc(F8C-EIA—C). The Bethesda titer of the four anti-FVIII ab against the two XTEN variants (pSD-088 and pSD-090) and the Refacto control were determined using d da assay methods,detailed as follows. Heat inactivated anti-FVIII dy samples at various ons were incubatedwith 1 IU/mL of each FVIII variant (diluted in 1X in FVIII chromogenic assay ) at a 1:1 ratio. Theantibody mixtures were then incubated for 2 hours in a 37°C incubator. After the incubation, thesamples were diluted for d with 1 x FVIII chromogenic assay buffer, and 25 [AL of diluted mixturewere then used for a FVIII chromogenic assay, The percentage of remaining FVIII activity wascalculated against the post-incubation activity of a known non-neutralizing sample. Bethesda units werecalculated using the following formula: BU=dilution factor X rcent of remaining activity) +6. 643 8).
: The results are listed in Table 47. Decreased Bethesda unit (BU) titers were observedfor all four antibodies when tested against the two FVIII-XTEN variants, in comparison with Refacto. Ato 8-fold fold decrease against PSD-088 and a 3 to 5-fold decrease against pSD-090, respectively, wereobtained. The inhibition curves against FVIII variants for each antibody were plotted () andcompared to Refacto, and demonstrates a clear left-shift of the inhibition curve for the two FVIII-XTENmolecules,with the pSD-O88 FVIII-XTEN variant ing in a further left-shift compared to pSD-O90.
These results clearly demonstrate that: 1) both FVIII-XTEN variant fusion proteins are more resistant topre-existing anti-FVIII inhibitory antibodies than Refacto; and 2) PSD-O88 is more resistant to anti-FVIIIantibodies than 0, which may provide information useful in determining the differences on theXTEN insertion sites in erring with the binding of anti-FVIII dies. Under the conditions ofthe experiment, the results provide some t for the potential use of FVIII-XTEN compositions fortreating hemophilia A patients with factor VIII inhibitors.
Table 47: Anti-FVIII antibod Bethesda titer a ainst FVIII-XTEN tsAnti-FVIII ab.04-483 05-505 GKl838-2079 F8C-EIA—C———“] Example 53: Half-life tions of FVIII XTEN fusion molecules containing fourXTEN insertions in Hemophilia A mice Methods: Eight FVIII-XTEN fusion proteins with four XTEN insertions each at definedlocations were tested in FVIII/VWF DKO mice to evaluate the effect of the XTEN insertions on FVIIIhalf-life extension: LSDOO71.00l, contains 403-AG144, l900-AE144, 745(B)-AE144, 2332(CT)-AE288XTEN insertions (designated as the FVIII amino acid number and the XTEN inserted); LSDOO71.002,containing 144, l900-AE144, -AE144, 2332(CT)-AE288 XTEN insertions;LSDOO72.001, containing 403-AG144, l900-AG144, 745(B)-AE144, 2332(CT)-AE288 XTENinsertions; LSDOO72.002, containing 403-AE144, l900-AG144, 745(B)-AE144, 2332(CT)-AE288XTEN insertions; pBCO247.004, containing l8-AG144, 403-AE144, l656-AG144, 2332(CT)-AE288XTEN insertions; pBCO251.002, containing l8-AG144, l656-AG144, l900-AE144, 2332(CT)-AE288XTEN insertions; pSDO88, containing 26-AG144, 403-AE144, l656)-AG144, E144 XTENinsertions and pSDO90, containing 44, l656-Agl44, l720-AG144, l900-AE144 XTENinsertions. FVIII/VWF DKO mice were treated, as generally described in Example 32, with a singleintravenous administration of FVIII-XTEN transfection cell media concentrate of the eight constructs at100-200 IU/kg, and plasma samples were subsequently collected at 5 min, 8 hrs, 24 hrs, 48 hrs, 72 hrsand 96 hrs post-dosing. Plasma FVIII actiVity was tested using the FVIII genic assay and FVIII-XTEN half-life was ted using the WinNonlin program.
RLults: All of the eight FVIII XTEN fusion molecules containing four XTEN ionsted longer half-life than unmodified FVIII (results in Table 48). Three molecules with XTENinsertions at positions 403, 1900, B domain, and C-terminal achieved half-life up to 16.3 hrs, which is ad improvement in comparison to unmodified BDD FVIII. However, the molecules tested withXTEN insertions at 26/403/1656/1900 8), or at 26/1656/1720/1900 (pSD090) showed ife of9.1 hrs and 9.5 hrs, respectively, Which, in comparison to BDD FVHI, represents an increase of 36-foldand 38-fold, respectively. pBC247.004 (XTEN insertions at 18/403/1656/CT) and pBC251.002 (XTENinsertions at 18/1900/1656/CT) achieved half-life values of 14.1 hrs and 13 hrs, respectively. The resultsdemonstrate that multiple XTEN ions (in this case, four XTEN insertions for each FVHI molecule)can significantly improve FVHI half-life. It further shows that the effect ofXTEN on FVHI half-life isinsertion site dependent, even in the event of multiple XTEN insertions.
Table 48: PK of FVIII-XTEN variants with four XTEN insertions in WF DKO miceent XTEN Insertions t1/2 (hr) t1/2 Increase (fold)BDD-FVIH None 0.25 NALSD0071 .001 1900AE/B/CT 16.2 64.8LSD0071.002 403AE/1900AE/B/CT 16.3 65.2LSD0072.001 403AG/1900AG/B/CT 11.8 47.2LSD0072.002 403AE/1900AG/B/CT 16.1 64.4ch247.004 18/403/1656/CT 14.1 56.4pBC251.002 18/1900/1656/CT 13.0 52pSD088 26/403/1656/1900 9.1 36.4pSD090 26/1656/1720/1900 9.5 38Table 49: Exem la Biolo icalActivi Exem la Assa s and Preferred IndicationsBiologically Active Exemplary ActivityProtein Biological Activity Assays Preferred Indication:Factor VIII Coagulation factor VIII is a Chromogenix assay Hemophilia A;(Factor VIII; factor essential for (Rosen 8, Scand J ng;Octocog alfa; hemostasis. This gene Haematol (1984) 33 Factor VIIIMoroctocog encodes coagulation (Suppl 40):139—45); deficiency;alfa; factor VIII, which participates Chromogenix bleeding esRecombinant in the intrinsic pathway of Coamatic® Factor VIII in patients withAntihemophilic blood coagulation; factor VIII assay; one-stage factor VIII inhibitor;factor; is a cofactor for factor lXa ng assay Surgeay~relatedNordiate; which, in the presence of Ca (Lethagen, 8., et al., hemorrhagico; + 2 and phospholipids, Scandinavian J esKogenate; converts factor X to the Haematology (1986)Kogenate activated form Xa. This gene 37:448—453.
SF; Helixate; es two alternatively One-stage clottingRecombinate) spliced transcripts. assay and two-stageTranscript variant l encodes clotting assaya large glycoprotein, isoform (Barrowcliffe TW,a, which circulates in plasma Semin Thromband associates with von Hemost. (2002)rand 28(3):247-256);factor in a noncovalent Development of acomplex. This protein simpleundergoes multiple chromogenic factor VIIIcleavage events. ript assay fort 2 encodes a putative clinical use.
PCT/U82012/046326Biologically Active Exemplary tyProtein Biological ty Assays red Indication:small protein, isoform b, (Wagenvoord RJ,which consists primarily of Hendrix HH,the olipid binding Hemker HC.domain of factor Vlllc. This Haemostasisbinding domain is essential 1989; 19(4): 196-204)for coagulant activity. Bethesda assayDefects in this gene results (Verbruggen B, et al.in hemophilia A, a common Improvements in factorrecessive X-linked VIII inhibitor ion:coagulation disorder. From Bethesda toNijmegen. SeminThromb . 2009Nov;35(8):752-759)Table 50: Exemplary CFXTEN comprising FVIII and internal/external XTEN seguences (SEQ IDNOS 1537-1554 res ectivel in order of a earanceCFXTENAmino Acid SequenceNameFVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(Al-K127 — AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAE144- REKEDDKGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEV128—N745- GSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGAE288- SEPATSGSETPGTSTEPSEGSAPGVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLP1640- VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAY2332) RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(Al-A375- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAE576- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRK376-N745- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNAE144- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDP1640- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD 2012/046326CFXTENAmino Acid SequenceNameY2332) DNSPSFIQIRSVAGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII BDD2 LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(A1-Y1792- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAF144- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRE1793- EKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNY2332- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDAE864) LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYGGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGCFXTENAmino Acid SequenceNameEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEFVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(A1-Y2043- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAG144- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRG2044- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNQ2222- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDAG864- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDV2223- DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYY2332) KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSGGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFN(A1-G1799- PWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSAE144- DKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVA1800- CREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYF2093- VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLAE42- LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV$2094- RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRV2223- IGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITAE42- SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDN2224- LASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEAE42- DPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYE 2012/046326CFXTENAmino Acid SequenceNameN2225- DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDG2278— ISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEAE42- DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSK2279- FTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGGGSEPY2332) ATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPGSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVGPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGGNNPKEWLQVDFQKTMKVTGVTTLTSMYVKEFLISSSQDGHQWTLFFQNGGTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARGPGSSPSASTGTGPGSSPSASTGTGPGTPGSG(Al-R28- TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSAG144-F29- PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSGFPPRVPKSFPFNTSVG244- VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYAG288- WKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLL245- VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR2090- RAWPKMHTVNGYVNRSLPGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPAG576- SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATQ2091- GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGY2332- SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSAAG864) GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAGINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGESTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEWO 22617 CFXTENAmino Acid SequenceNameGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI(Al-T1651- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAG576- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRR1652- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNK1808- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDAG144- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDP1809- DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYF2093- KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLAG288- YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIS2094- GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAY2332) SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSSGRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSCFXTENAmino Acid SequenceNameASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDAGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSG(Al-A28- ATGSPGPSGPGRFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVAG42-F29- YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEGGPGTPGSGTASSSPGE124- SSTPSGATGSPGSSPSASTGTGPGASPGDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSAG42- LVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGGSPSASTGTGPGASD125-E124- PGTSSTGSPGTPGSGTASSSPGSSTPSGAGKSWHSETKNSLMQDRDAASARAWPKMHTVNAG42- GYVNSSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTD125-P333- QFLLFCHISSHQHDGMEAYVKVDSCPEEPGSASTGTGPGASPGTSSTGSPGTPGSGAG42- TASSSPGSSTPSGATGGQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKQ334- HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFY2332) KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWTPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI(Al-D345- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAE144- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRY346- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVND403- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDAE144- FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDGGSEPATSGSETPGTSESR405- ATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSER1797- TPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGYDAE288- DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDGGTQ1798- SASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAEY2322) SPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLCFXTENAmino Acid SequenceNameSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWARLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII (A1- ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIN745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAE864- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR(P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNY2332) IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTS V V Y KKTLFVEFTVHLFNI(Al- N745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAE288- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRCFXTENAmino Acid SequenceName(P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNY2332) RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQFVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(Al- S743)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAE288- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR(Q1638- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNY2332) RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQFVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(Al- N745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAG288_2- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR(P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNY2332)- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDCFXTENAmino Acid SequenceName2 LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPFVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI(Al- S743)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQAG288_2- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR(Q1638- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDAG288_2 LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTCFXTENAmino Acid SequenceNameGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIBDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ(Al- N745)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRAE288- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN(P1640- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDY2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDAE288 IQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIBDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ(Al- S743)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRAE288- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN(Q1638- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDY2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDAE288 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTCFXTENAmino Acid SequenceNameQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIBDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ(Al- N745)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRAG288_2- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN(P1640- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDY2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDAG288_2 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIBDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ(Al- S743)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCRAG288_2- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN(Q1638- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDAG288_2 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQNPPVLKRHQAEITRTTLQSDWO 22617 CFXTENAmino Acid SequenceNameQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATce name reflects N— to C-terminus configuration of the FVIH segments (amino acid spanningnumbers relative to mature sequence) and XTEN componentsTable 51: Exemplary CFXTEN comprising FVIII, cleavage seguences and XTEN ces (SEQID NOS 1555-1590 res ectivel in order of a earanceCFXTENAmino Acid SequenceNameSP-AE288- MQIELSTCFFLCLLRFCFSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPCS-L- GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA(FVIII_1- TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP745)— GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSAE288- EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQGPEGPSATRRYYLGAVE(FVIII_168 LSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLG6-2332)-L- PTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHCS-AE288 TYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSlNGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSlNAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVCFXTENAmino Acid SequenceNameTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSSP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEECS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS(FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP745)— GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSAE576- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG(FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE6-2332)-L- GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSCS-AE288 EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQGPSGPATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYRLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSP- TCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV(FVIII_1- VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW745)— KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKAE576- DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW(FVIII_168 NGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI)-L- TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDCS-AE576 SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHWO 22617 CFXTENAmino Acid SequenceNameGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPTPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVELHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPSP-AE576- TCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEECS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS(FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP745)— GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSAE576- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG(FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE6-2332) GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQGPEGPSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGEGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSCFXTENAmino Acid SequenceNameEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYSP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEECS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS(FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP743)— GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSAE288- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE)-L- GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS76 EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPIEPRSPSGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSSATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGIEPRSPSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPSP-AG288- MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGCFXTENAmino Acid SequenceNameCS-L- SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP(FVIII_1- SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT743)— GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPAG576- GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSIEPRSPSGSPGATRRYYLGAVEL(FVIII_168 SWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGP6-2332)-L- TIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT88 YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSSP-AG576- MQIELSTCFFLCLLRFCFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTCS-L- GPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG(FVIII_1- SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS745)— GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPAG288- GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT(FVIII_168 GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP6-2332)-L- GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGCS-AE576 SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGECFXTENAmino Acid SequenceNameTVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSEGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPSP- MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV_1- FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW743)— EYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKAG576- IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW(FVIII_168 NGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI6-2332)-L- TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDCS-AG576 SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGCFXTENAmino Acid SequenceNameATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSSP-AG288- MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGCS-L- SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP_1- SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT743)— GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPAG288- GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQGPSGPATRRYYLGAV(FVIII_168 ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLL6-2332)-L- GPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSCS-AE288 HTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGETVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSL1SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPSGPQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEECS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS(FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP743)— GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSAG576- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG(FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE6-2332) GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI 2012/046326CFXTENAmino Acid SequenceNameCYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSL1SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDDZ KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKS3 67- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLFXIa- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNSSLPGLAE42- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFF3 68- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSKLTY23 32- RAETGEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSGFIQIRSVAKKHPKTWVHYFXIa- IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGAE864 ILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSL1SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWTPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPCFXTENAmino Acid SequenceNameFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDDZ KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKN745 - EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLFIXa- TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLAGZ88- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFFIXa- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIP 1 640- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYY23 32- TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKFIXa- HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDAG864 QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPLGRIVGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGPLGRIVGGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENNGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYPLGRIVGGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDDZ KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKV1 2 8- EDDKVLQVRIVGGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGPGLQVRIVGGFVIIa- FPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQAG42- TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKFVIIa- SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHG2044- AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKFVIIa- KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFAG144- QHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFY23 32- PILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQFVIIa- NVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVAG576 CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPCFXTENAmino Acid SequenceNameLGMASGHIRDFQITASGQYGLQVRIVGGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGLQVRIVGGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYLQVRIVGGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSAE864- GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSFVIII- EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGThrombin- TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPAE144 ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEELESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFCFXTENAmino Acid SequenceNameDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDD3 - KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGTMTRIVGGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDD3 - KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKElastase- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPWO 22617 CFXTENAmino Acid SequenceNameGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDD3 - MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEALTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESATSGSETPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIABDD3 - KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKThrombin- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPAE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATFVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPBDDZ- GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTMMP- 1 7- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE864 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTCFXTENAmino Acid SequenceNameVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAPLGLRLRGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATFVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPBDDZ- GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTFXIIa- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE864 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLCYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGTMTRIVGGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSECFXTENAmino Acid SequenceNameTPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAG144- SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTFVIII GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPBDDZ- SASTGTGPGASPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTFXIa- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAG576 TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGKLTRAETGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSAE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATFXIa-FVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPBDDZ- SEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTAE864 TLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDCFXTENAmino Acid SequenceNameTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATFVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPBDDZ- GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTY23 32- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGThrombin- TSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLAE864 IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGLTPRSLLVGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSACFXTENAmino Acid SequenceNamePGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAE864- GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSFVIII- EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGMMP TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPAE144 ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGAPLGLRLRGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPAF144- GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTACFXTENAmino Acid SequenceNameFXIIa- ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTFVIII- SPSGESSTAPGTMTRIVGGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVFXIIa- VYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWAF864 KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVSSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLHPQSWVHQIALRMEVLGCEAQDLYGTMTRIVGGGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTATSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPAE864- GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSFVIII- EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGFXIa- TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPAE144 ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPACFXTENAmino Acid SequenceNameGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGPESGPGSEPATSGSETPGTSTEPSEGSAPAE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATFXIa-FVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPBDD9- SEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTAE864 SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQCFXTENAmino Acid SequenceNameSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLPEQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPAE48— MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGKLTRAETGATRRYYFXIa-FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWBDD9- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFAE864 PGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTWO 22617 CFXTENAmino Acid ceNameEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD9- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAG288_2 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD9- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAG864 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSWO 22617 CFXTENAmino Acid SequenceNamePGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD9 (1— KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK745) EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAG288_2- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL(1640- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF- QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIFXIa- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYAG864 TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMEAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD9 (1— KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK743) EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAG288_2- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL(163 8- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFY2332)- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIFXIa- HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYAG864 TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGCFXTENAmino Acid SequenceNameSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPBDD10 (1— ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA745) KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKAG288_2- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL(1640- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLY2332)- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFFXIa- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIAG864 RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPCFXTENAmino Acid SequenceNameGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD10- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAG288_2 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEATRAETGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATGFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD 1 0- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAG864 TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEACFXTENAmino Acid SequenceNameQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPFVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIABDD l 0- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKFXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAE864 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSequence name reflects N— to C-terminus configuration of the FVIII variant and XTEN components:signal peptide (SP); linker (L); cleavage sequence (CS) may be denoted by protease name active on thesequence, and XTEN components by family name and length, With insertion points for componentsdenoted by FVIII amino acid and numbered positions adjacent to the inserted sequence or Al being theN—terminus and Y2332 being the C-terminus of the FVIII.
The term “comprise” and variants of the term such as “comprises” or “comprising” are usedherein to denote the inclusion of a stated integer or stated integers but not to excludeany other integer orany other integers, unless in the context or usage an exclusive interpretation of the term is ed.
Any reference to publications cited in this cation is not an admission that thedisclosures constitute common general knowledge in Australia.309a