The application is international application no PCT/EP2012/055830, international filing date 2012-03-30, China's application number201280016811.X, China's day 2013-09-30, invention entitled " use treats immunity for the single domain antibody of TNF αThe method of disease " divisional application.
Accompanying drawing describes
Fig. 1 shows that reached rheumatoid in being administered the often group of 6 treatment groups of ozoralizumab closed at the 8th and 16 weekThe ACR-20 of joint inflammation treatment, 50 and 70 patient's percent of standard.In the dosage regimen of every 4 weeks, experimenter the 1st day,4, within 8 and 12 weeks, it is administered ozoralizumab or placebo.In the scheme of every 8 weeks, experimenter was administered at the 1st day and the 8th weekOzoralizumab, and used placebo at the 4th and 12 week.
Fig. 2 shows treatment group ACR 20 responsiveness (LOCF) in time.
Fig. 3 shows treatment group DAS28 responsiveness (LOCF) in time.
Fig. 4 shows the dosage-ACR20 response of intermediate value (95%PI) placebo-correction of ATN-103.Shown in dotted line showsComparison therapy intermediate value simulation response.
Fig. 5 shows the DAS response that the intermediate value (95%PI) of ATN-103 is simulated.Dotted line show shown in comparison therapy inValue response.
Fig. 6 shows the medicinal effectiveness represented with %ACR20 and %SI.Each bubble covers every kind of medicine in shown dosage regimenThe 95%PI of analog response.
Definition
In order to the present invention is more easily understood, first define some term.Additionally be defined on whole detailed descriptionMiddle elaboration.
When used herein, article " " and " a kind of " (" a " and " an ") refer to that one or more than one is (such as, extremelyFew one) grammatical object of this article.
Term "or" is with being here and hereinafter meant that term "and/or", and can exchange use with it, unless context is additionallyClearly indicate.
Term " albumen " and " polypeptide " are used interchangeably herein, and include the SDAB molecule of the present invention.
" about " and " about " generally means that acceptable about measured quantity providing the character of measurement or degree of accuracyError degree.Exemplary error degree is within 20 percent (%) of given numerical value or numerical range, typicallyWithin 10%, more typically within 5%.
When modify term such as " about ", " about ", " at least " or " at most " before a series of terms time, this term is intended toEach in term listed by modification.Such as, phrase " at least about 10% or 20% " should be construed to " at least about 10% or extremelyFew about 20% ".
In the situation of nucleotide sequence, term " substantially the same " includes foot with in this article referring to the first nucleotide sequenceEnough or the minimal number of nucleotide identical with the nucleotide of comparison in the second nucleotide sequence, so that the first and second nucleotides sequencesRow coding has the polypeptide of common functional activity, or encodes common structure polypeptide domain or common functional polypeptide activity, exampleAs, with canonical sequence have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% orThe nucleotide sequence of 99% homogeneity.
The present invention also includes the fragment of polypeptide, derivant, analog or the variant of the present invention, and their any groupClose.Term " fragment ", " variant ", " derivant " and " analog " includes retaining corresponding natural when referring to the albumen of the present inventionAny polypeptide of at least some functional characteristic of SDAB molecule.The specific Fab discussed except elsewhere hereinOutside, the fragment of the polypeptide of the present invention also includes hydrolysis and deletion fragment.The variant of the polypeptide of the present invention includes as described aboveFragment, and also include due to aminoacid replacement, the polypeptide that lacks or insert the aminoacid sequence with change.Variant is permissibleIt is naturally-occurring or non-naturally-occurring.The variant of non-naturally-occurring can use induced-mutation technique known in the art to produce.BecomeBody polypeptide can include that conservative or nonconserved amino acid replaces, lacks or add.The derivant of the fragment of the present invention is suchPolypeptide, described polypeptide be changed thus show natural polypeptides non-existent other feature.Example includes fusion protein.Variant polypeptide can also be referred to as " polypeptide analog " in this article.When used herein, " derivant " of polypeptide refers to have logicalCross the reaction of functional side group's group and the desired polypeptides of chemically derived one or more residues." derivant " also includes containing oneThe those polypeptides of the naturally occurring amino acid derivativges of individual or multiple 20 kinds of standard amino acids.Such as, 4-hydroxyprolineProline can be replaced；5-oxylysine can replace lysine；3-Methyl histidine can replace histidine；HomoserineSerine can be replaced；Ornithine can replace lysine.
Term " functional variety " refers to have substantially the same aminoacid sequence with naturally occurring sequence or by substantiallyUpper the most identical nucleotide sequence coded and can have the polypeptide of one or more activity of naturally occurring sequence.
Homology between sequence or the calculating of sequence iden (this term is used interchangeably herein) by following enterOK.
In order to determine the homogeneity percent of two kinds of aminoacid sequences or two kinds of nucleotide sequences, described sequence is for most preferably to comparePurpose is compared (for example, it is possible to introduce breach in the one or both of the first and second aminoacid or nucleotide sequence, to enterThe optimal comparison of row, and for comparative purposes, non-homology sequence can be ignored).In a typical implementation, for comparingThe length of the canonical sequence of purpose comparison is at least the 30% of the length of described canonical sequence, 40%, 50%, 60%, 70%,80%, 90% or 100%.
Then compare at corresponding amino acid position or the amino acid residue of nucleotide position or nucleotide.When firstWhen position in sequence is occupied by the identical amino acid residue in the position corresponding with the second sequence or nucleotide, then two kinds pointsSon in this position be identical (when with time in this article, aminoacid or nucleic acid " homogeneity " and aminoacid or nucleic acid " homology "Of equal value).
In view of the length (needing to introduce described breach with optimal comparison both sequences) of breach number and each breach,Homogeneity percent between two kinds of sequences is the function of the number of the same position that described sequence has.
The determination of the homogeneity percent between the comparison of sequence and two kinds of sequences can use mathematical algorithm to realize.OneIn individual embodiment, the homogeneity percent between two kinds of aminoacid sequences uses Needleman&Wunsch, J.Mol.Biol.Algorithm described in (J. Mol. BioL) 48:444-453 (1970) determines, this algorithm has been incorporated into GCG software kitGAP program in (on the Internet gcg.com available), it uses Blossum 62 matrix or PAM250 matrix, and 16,14,The Gap Weight of 12,10,8,6 or 4 and the Length Weight of 1,2,3,4,5 or 6.In another embodiment, two kinds of nucleotideHomogeneity percent between sequence uses the GAP program in GCG software kit to determine (available on the Internet gcg.com), itsUse NWSgapdna.CMP matrix and the Gap Weight of 40,50,60,70 or 80 and the Length Weight of 1,2,3,4,5 or 6.One groupTypical parameter (and the parameter that should use except as otherwise noted) is Blossum 62 rating matrix, and it has Gap Penalty12, gap extension penalty 4, and frameshift gap point penalty 5.
Homogeneity percent between two kinds of aminoacid or nucleotide sequence can use E.Meyers and W.MillerAlgorithm described in CABIO, 4:11-17 (1989) determines, this algorithm is already integrated in ALIGN program (version 2.0)In, it uses PAMl20 weight residue table, Gap Length Penalty 12 and Gap Penalty 4.
Nucleic acid as herein described and protein sequence can serve as " search sequence " and retrieve public database, exampleAs, thus identify other family members or relevant sequence.Such retrieval can use Altschul et al..J.Mol.Biol. NBLAST and the XBLAST program (version 2.0) of (J. Mol. BioL) 215:403-10 (1990)Carry out.NBLAST program can be used to carry out BLAST nucleotide search, scoring=100, word length=12, thus obtain and thisThe nucleotide sequence of the nucleic acid molecule homologous described in bright.XBLAST program can be used to carry out BLAST Protein hits, mark=50, word length=3, thus obtain and the aminoacid sequence of albumen of the present invention (SEQ ID NO:1) molecule homologous.For terribleTo comparison jaggy for comparative purposes, it is possible to use BLAST jaggy (Gapped BLAST), as at AltschulEt al., described in Nucleic Acids Res. (nucleic acids research) 25:3389-253402 (1997).When using BLAST and breachDuring the blast program changed, it is possible to use the default parameters of described various programs (such as, XBLAST and NBLAST).
" conserved amino acid replacement " is that wherein amino acid residue is had the radical amino acid replacement of similar side chain.In abilityTerritory has been defined for the family with the amino acid residue of similar side chain.These families include the aminoacid with basic side chain(such as, lysine, arginine, histidine), has the aminoacid (such as, aspartic acid, glutamic acid) of acid side-chain, has notThe aminoacid of charged polar side chain (such as, glycine, agedoite, glutamine, serine, threonine, tyrosine,Cysteine), have non-polar sidechain aminoacid (such as, alanine, valine, leucine, isoleucine, proline, benzeneAlanine, methionine, tryptophan), there is the aminoacid (such as, threonine, valine, isoleucine) of the side chain of β-side chainAnd there is the aminoacid (such as, tyrosine, phenylalanine, tryptophan, histidine) of beta-branched side.
Term " SDAB " refers to that the single domain antigen as described in W004/041862 and WO 06/122786 combines and dividesSon.Term " SDAB molecule " refers to polypeptide or other molecules of one or more SDABs.Term " SDAB " and " SDAB dividesSon " use can be exchanged, represent that only domain antigen-binding units (such as, TNF30 or ALB8) or multivalent single domain resistFormer binding structural domain (such as, TNF55, TNF56 or orozoralizumab).
" CDR " of variable domains is cumulative, the AbM according to both Kabat, Chothia, Kabat and Chothia, connectsTouch (contact) and/or conformation definition or any CDR well known in the art determines that the aminoacid in the hypervariable region that method is identified is residualBase.Antibody CDRs can be accredited as the hypervariable region initially defined by Kabat et al..For example, with reference to Kabat et al., 1992,Sequences of Proteins of Immunological Interest (protein sequence that immunity is interested), the 5th edition,Public Health Service, NIH, Washington D.C..The position of CDRs can also be accredited as initially by ChothiaWith the structure ring structure other people described.For example, with reference to Chothia et al., 1989, Nature (naturally) 342:877-883.ItsHe identifies that the method for CDR includes " AbM definition ", and it is the half-way house between Kabat and Chothia, and uses OxfordMolecular ' s AbM antibody modeling software (is now) derivative, or it is included in MacCallum et al., 1996,J.Mol.Biol. (J. Mol. BioL), described in 262:732-745 based on viewed antigen contact(contact) " the contact definition " of CDRs.In another approach, " the conformation definition " of herein referred as CDRs, the position of CDRsPut to be accredited as and antigen is combined the residue making enthalpy contribution.For example, with reference to Makabe et al., 2008, JournalOfBiological Chemistry (journal of biological chemistry), 283:1156-1166.Also other CDR boundary definitions can notIn strict accordance with one of said method, but will at least some of overlapping, although according to specific residue with Kabat CDRsOr organize residues or the prediction of the most whole CDRs not appreciable impact antigen combination or experiment discovery more, it can be shortened or addLong.When used herein, CDR may refer to be defined by method as known in the art (including the combination of various method)CDRs.Method used herein can use the CDRs according to any one definition in these methods.For the bag be arbitrarily givenInclude the embodiment of more than one CDR, CDRs can according to Kabat, Chothia, extension, AbM, contact and/or conformation fixedAny one definition in justice.
Detailed Description Of The Invention
Single domain antigen combines (SDAB) molecule
Single domain antigen combines (SDAB) molecule and includes such molecule, and its complementary determining region is single domain polypeptideA part.Example includes, but not limited to heavy-chain variable domains, the binding molecule of natural shortage light chain, derives from conventional 4-chainThe single domain of antibody, the domain of transformation and the single domain framework being different from the framework deriving from antibody.SDAB molecule canTo be any single domain molecule or the single domain molecule in any future of this area.SDAB molecule can derive from anySpecies, include, but not limited to mice, people, camel, yamma, fish, shark, goat, rabbit and cattle.
According to an aspect, the single domain antigen binding molecules that SDAB molecule is naturally-occurring, it is known as lacking gentlyThe heavy chain of chain.Such as, described single domain molecule is disclosed in WO 94/04678 and Hamers-Casterman et al. .NatureIn (naturally) 363:446-448 (1993).For clearly reason, this derives from heavy chain molecule variable of natural shortage light chainDomain is properly termed as VHH, to distinguish the conventional VH of itself and four chain immunoglobulins.Described VHH molecule can derive from camelSection's species (Camelidae species), such as, camel (camel), yamma (llama), dromedary camel (dromedary),Alpaca (alpaca) and maroon alpaca (guanaco).It is light that other species in addition to camellid can produce natural shortageThe heavy chain molecule of chain；This type of VHHs is within the scope of the present invention.
Described SDAB molecule can be restructuring, CDR-grafting, humanized, camelized, go immunity and/or(such as, selected by phage display) of external generation, as described in greater detail below.
Term " antigen-combination " is intended to include the part of polypeptide, such as, the part of single domain molecule as herein described,It determinant including forming the interface combined with target antigen (or its epi-position).About albumen (or albumen analogies), antigen-knotClose site and typically comprise one or more rings (at least four aminoacid or the amino Radix rumicis acetosae forming the interface being combined with target antigenIntend the ring of thing).Typically, the antigen binding site of polypeptide, such as, the antigen binding site of described single domain antibody molecule,Including at least one or two CDRs, or more typically include at least three, four, five or six CDRs.
Term " immunoglobulin variable domain territory " is generally understood as and VL or VH in human or animal source in the artDomain is identical or substantially the same.It should be appreciated that in some species, such as, in shark and yamma, immune globulinWhite variable domains can be evolved, thus different from VL or VH of people or mammal on aminoacid sequence.
But, these domains are still primarily involved in antigen and combine.Term 20 " immunoglobulin variable domain territory " typical caseGround includes at least one or two CDRs, or more typically at least three CDRs.
" constant immunoglobulin domains " or " constant region " are intended to include and CL, CH1, the CH2 in human or animal source,The immunoglobulin domains that CH3 or CH4 domain is identical or essentially similar.For example, with reference to, Hasemann and Capra,Immunoglobulins:Structure and Function (immunoglobulin: 26S Proteasome Structure and Function), at WilliamE.Paul compiles, Fundamental Immunology (basic immunology), the second edition, 209,210-218 (, 1989) in.Term" Fc district " refers to the Fc part of constant immunoglobulin domains, it include immunoglobulin domains CH2 and CH3 or and theseEssentially similar immunoglobulin domains.
In certain embodiments, described SDAB molecule be unit price or multispecific molecule (such as, bivalence, trivalent orTetravalent molecule).In other embodiments, SDAB molecule is specific point of monospecific, bispecific, tri-specific or fourSon.Molecule is " monospecific " or " polyspecific ", e.g. " bispecific ", refers to that Binding peptide reactsThe number of different epi-positions.Multispecific molecule can be the different epitope specificities to target polypeptide as herein described, orCan be to target polypeptid specificity and specific to heterologous epitope (such as heterologous polypeptide or solid support matter).
When used herein, term " valency " refers to the number of binding structural domain potential present in the SDAB molecule, exampleAs, the number of antigen-binding domains.The specific binding epi-position of each binding structural domain.When SDAB molecule includes more than oneDuring individual binding structural domain, each binding structural domain can be with specific binding identical epi-position, for having two basic change domainAntibody, be referred to as " bivalence monospecific ", or can be with specific binding different epi-position, for having two basic change domainSDAB molecule, be referred to as " bivalent, bispecific ".SDAB molecule can also be bispecific and for every species specificity two(referred to as " the bispecific tetravalent molecule ") of valency.Bispecific bivalent molecule, and the method preparing it, such as, describe in the U.S.The patent No. 5,731,168；5,807,706；In 5,821,333；With U.S. Publication No 2003/020734 and 2002/0155537In；All these disclosures is incorporated herein by reference.Bispecific tetravalent molecule, and the method preparing it, such as, noteStating in WO 02/096948 and WO 00/44788, the disclosure of the two is incorporated herein by reference.Polyspecific and multivalenceOther examples of molecule provide at WO 93/17715；WO92/08802；WO 91/00360；WO 92/05793；U.S. Patent number4,474,893；4,714,681；4,925,648；5,573,920；With 5,601,819；Tutt et al., J.Immunol. (immunityLearn magazine) 147:60-69 (1991)；With Kostelny et al., J.Immunol. (Journal of Immunology) 148:1547-1553(1992) in.
In certain embodiments, SDAB molecule is the single chain fusion polypeptide combining one or more target antigens, and it includesThe complementary variable domains of one or more shortages or the single domain molecule of constant region for immunoglobulin (such as, Fc district).By instituteThe exemplary target antigen stating antigen-binding polypeptide identification includes tumor necrosis factor α (TNFa).In specific embodimentIn, described Antigen-Binding single domain molecule combines serum albumin, such as, in conjunction with selected from serum albumin (such as, human bloodPure albumen (HSA)) or the human albumin of one or more of transferrins.
TNFa
It is known in the art tumor necrosis factor α (TNFa) relevant to inflammatory disease, described inflammatory disease such as class windWet arthritis, Crohn disease, ulcerative colitis and multiple sclerosis.Have studied TNFa and receptor the most in detail(CD120a and CD120b).The biologically active form of TNFa is trimer.Have been developed for some and use anti-TNF a Antibodies AgainstThe strategy of TNFa effect, and be currently and can be purchased, such asWithUnijunction in conjunction with TNFaThe multiple example of structure territory antigen binding molecules is disclosed in WO 04/041862, WO04/041865, in WO 06/122786, allThese content is fully incorporated in this by quoting.
The other example of single domain antigen binding molecules is disclosed in US 2006/286066, US2008/0260757,In WO 06/003388, US 2005/0271663 and US 2006/0106203, all these contents is tied completely by quotingTogether in this.In particular embodiments, the SDAB molecule of the present invention includes the SDABs of the aminoacid sequence of table 1, or bagInclude the aminoacid sequence that includes having the sequence iden of about 80%, 85%, 90%, 95% or 99% with the sequence of table 1SDABs.In alternate embodiment, the SDAB molecule of the present invention can include having 1 with the sequence of table 1,2,3,4,5,6,The aminoacid sequence of 7,8,9,10,11 or 12 aminoacid differences.
In other embodiments, it is considered to for TNFa and the monospecific of serum albumin (such as HSA), bispecific,Tri-specific and other polyspecific single domain antibodies.The multiple example of polyspecific TNFa and HSA Binding peptide is disclosed inWO 06/122786 kind, its content is incorporated herein.The polyspecific polypeptide of the present invention can include the ammonia of table 1Base acid sequence, or include there is the aminoacid sequence of about 80%, 85%, 90%, 95% or 99% sequence iden with the sequence of table 1Row.In alternate embodiment, the polyspecific SDAB molecule of the present invention can include having 1 with the sequence of table 1,2,3,4,The aminoacid sequence of 5,6,7,8,9,10,11 or 12 aminoacid differences.
In particular embodiments, the SDAB molecule in conjunction with TNFa includes disclosed in one or more WO06/122786SDABs.Such as, the SDAB molecule of described combination TNFa can be the unit price disclosed in WO 06/122786, bivalence or trivalentTNFa-Binding peptide.
The SDABs molecule of the exemplary combination TNFa disclosed in WO 06/122786 includes, but not limited to TNF1,TNF2, TNF3, its humanization form (such as, TNF29, TNF30, TNF31, TNF32, TNF33).The table of WO 2006/122786The other example that unit price combines the SDABs of TNFa is disclosed in 8.Exemplary bivalence combines the SDAB molecule of TNFa and includes,But being not limited to, TNF55 and TNF56, it includes two TNF30SDABs connected by peptide linker, thus forms single fused polypeptide(disclosed in WO 06/122786).Bivalence combines the other example table 19 at WO 06/122786 of the SDAB molecule of TNFaDisclosed in be TNF4, TNF5, TNF6, TNF7, TNF8.
In particular embodiments, anti-TNF a SDAB includes 3 complementary determining regions (CDR1, CDR2 and CDR3), itsIn: CDR1 includes aminoacid sequence DYWMY (SEQ ID NO:22)；With DYWMY (SEQ ID NO:22), there is at least 80% sequenceThe aminoacid sequence of row homogeneity；Or with DYWMY (SEQ ID25 NO:22), only there is the aminoacid sequence of 1 aminoacid differenceRow；CDR2 includes aminoacid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23)；With EINTNGLITKYPDSVKG (SEQID NO:23) have at least 80%, the aminoacid sequence of 90% or 95% sequence iden；Or and EINTNGLITKYPDSVKG(SEQ ID NO:23) has the aminoacid sequence of 2 or 1 aminoacid differences；And CDR3 includes aminoacid sequence SPSGFN(SEQ ID NO:24)；With the aminoacid sequence that SPSGFN (SEQ ID NO:24) has at least 80% sequence iden；Or withSPSGFN (SEQ ID NO:24) has the aminoacid sequence of 1 aminoacid difference.
In some embodiments, include the aminoacid sequence of table 1 in conjunction with the SDAB of HSA, or have about with the sequence of table 1The aminoacid sequence of 80%, 85%, 90%, 95% or 99% sequence iden.In alternate embodiment, the present invention'sAnti-HSA SDAB can include having the ammonia of 1,2,3,4,5,6,7,8,9,10,11 or 12 aminoacid difference with the sequence of table 1Base acid sequence.
In other embodiments, the SDAB molecule in conjunction with HSA includes one or more disclosed in WO 06/122786SDABs.Such as, the SDAB molecule of described combination HSA can be that the unit price disclosed in WO 06/122786, bivalence or trivalent combineThe SDAB molecule of HSA.In other embodiments, the SDAB molecule of described combination HSA can be to have at least one to combine HSAThe monospecific of binding specificity or multispecific molecule.The SDABs of exemplary combination HSA includes, but not limited to WOALB1 disclosed in 06/122786 and humanization form (such as, ALB6, ALB7, ALB8, ALB9, ALB10) thereof.
In particular embodiments, described anti-HSA SDAB includes 3 CDRs (CDR1, CDR2 and CDR3), wherein:CDR1 includes aminoacid sequence SFGMS (SEQ ID NO:25；There is at least 80% sequence together with SFGMS (SEQ ID NO:25)The aminoacid sequence of one property；Or with SFGMS (SEQ ID NO:25), only there is the aminoacid sequence of 1 aminoacid difference；CDR2Including amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26)；With SISGSGSDTLYADSVKG (SEQ ID NO:26) have at least 80%, the aminoacid sequence of 90% or 95% sequence iden；Or with SISGSGSDTLYADSVKG (SEQ IDNO:26) there is the aminoacid sequence of 2 or 1 aminoacid differences；And CDR3 includes aminoacid sequence GGSLSR (SEQ IDNO:27)；With the aminoacid sequence that GGSLSR (SEQ ID NO:27) has at least 80% sequence iden；Or and GGSLSR(SEQ ID NO:27) has the aminoacid sequence of 1 aminoacid difference.
In other embodiments, two or more single domain molecules of described SDAB molecule are with or without linkerGroup is fused to heredity or polypeptide fusion.Described linking group can be any linking group that those skilled in the art know.Such as, described linking group can be the biocompatible polymer of a length of 1 to 100 atom.In one embodiment,Described linking group includes following or is made up of following: polyglycine, polyserine, polylysine, polyglutamine, poly-different brightPropylhomoserin or poly arginine residue or combinations thereof.Such as, polyglycine or polyserine linking group can include at least fiveIndividual, seven, eight, nine, ten, 12,15,20,30,35 and 40 glycine and silkHistidine residue.The exemplary joint that can use includes that Gly-Ser repeats, such as, with one, two, three, four, fiveIndividual, six, seven or (Gly) of more repetition4-Ser (SEQ ID NO:19) repeats.In some embodiments, jointThere is following sequence: (Gly)4-Ser-(Gly)3-Ser (SEQ ID NO:20) or ((Gly)4-Ser) n (SEQ ID NO:21),Wherein n is 4,5 or 6.In some embodiments, joint includes SEQ ID NO:6 (GS9) or SEQ ID NO:7 (GS30).
In an exemplary embodiment, the SDAB molecule of the present invention can by two basic change target antigen (such asTNFa) SDAB domain (such as, two camellid variable regions) and a unijunction combining serum albumin (such as HSA)The single-chain polypeptide fusion body of structure domain antibodies molecule (such as, camellid variable region) is constituted.
Being referred to herein as the polypeptide of " ozoralizumab " is melting of humanized, trivalent, bispecific suppression TNFaHop protein.This fusion protein derives from camellid and has sequence and the knot of height with human normal immunoglobulin's VH domainStructure homology.Its Single polypeptide chain is made up of two binding structural domains for TNFa and a binding structural domain for HSA,Two nine amino acid whose G-S joints are utilized to connect described domain.The detailed description of Ozoralizumab provides at WO 06/In 122786 (wherein it is referred to as TNF60), its content is incorporated herein.
Table 1
The preparation of SDAB molecule
The SDAB molecule of the present invention can include that one or more are recombinated, CDR-grafting, humanized, camelized, remove immunity and/or external generation (such as, being selected) single domain molecule (such as SDABs) by phage display.The technology producing antibody and the technology of SDAB molecule and these molecules of recombinant modified is well known in the art, and belowDescribe in detail.
Multiple method well known by persons skilled in the art may be used for obtaining antibody.Such as, monoclonal antibody can be passed throughProduce hybridoma according to known methods and prepare.Then, use standard method, such as enzyme-linked immunosorbent assay (ELISA) andSurface plasma body resonant vibration (BIACORETM) hybridoma that formed by this way of Analysis and Screening, thus identify generation specificity knotClose one or more hybridomas of the SDAB of the antigen specified.Any type of antigen specified can serve as immunogen, such as,Recombinant antigen, naturally occurring form, its arbitrary variant or fragment, and its antigenic peptides.
A kind of exemplary method preparing antibody and SDAB molecule includes screening protein expression libraries, such as, phageOr ribosomal-display library.Such as, phage display describes at U.S. Patent number 5,223,409；Smith Science 228:1315-1317(1985)；WO 92/18619；WO 91/17271；WO 92/20791；WO 92/15679；WO 93/01288；WO 92/01047；WO 92/09690；With in WO 90/02809.
Except using in addition to display libraries, it is intended that antigen can be used to immunizing non-human animals, such as, rodent, exampleAs, mice, hamster or rat.In one embodiment, described non-human animal includes at least some of human normal immunoglobulin's baseCause.For example, it is possible to the large fragment of employment Ig locus is transformed lacks the mouse species that mouse antibodies produces.Use hybridoma skillArt, can produce and select have the specific antigenic specificity monoclonal antibody deriving from gene in need.For example, with reference toXENOMOUSETM, Green et al. .Nature Genetics (natural genetics) 7:13-21 (1994), US20030070185,WO 96/34096 and PCT Application No. PCT/US96/05928.
In another embodiment, SDAB molecule is obtained by non-human animal, is then modified, and such as, humanization, goes to exempt fromEpidemic disease and/or chimeric, it is possible to use recombinant DNA technology known in the art produces these SDAB molecules.Describe multipleFor preparing chimeric antibody and the method for SDAB molecule.For example, with reference to Morrison et al.,Proc.Natl.Acad.Sci.U.S.A. (NAS's journal) 81:6851 (1985)；Takeda et al., Nature(naturally) 314:452 (1985), U.S. Patent number 4,816,567 and 4,816,397；European Patent Publication EP171496 and0173494；And British patent GB 2177096B.For example, it is also possible to but use is expressed human immunoglobulin gene can not tableReach the SDAB molecule that the transgenic mice of endogenous mouse immunoglobulin gene is generating humanized.Winter describes a kind of exampleThe CDR-engrafting method of property, its can be used to prepare humanized SDAB molecule as herein described (U.S. Patent number 5,225,539).All CDRs of specific SDAB molecule by least some of replacement of inhuman CDR, or can only have some CDRsCan be replaced by inhuman CDRs.Only need to replace described SDAB molecule and be combined required number with predetermined antigenCDRs。
Humanized SDAB molecule the most directly can participate in resisting by replacing with the equivalent sequence from people's variable domainsThe sequence of the variable domains of former combination and produce.The exemplary method of generating humanized antibodies or its fragment is by MorrisonScience (science) 229:1202-1207 (1985)；Oi et al., BioTechniques (biotechnology) 4:214 (1986)；WithU.S. Patent number 5,585,089；5,693,761；5,693,762；5,859,205；There is provided with 6,407,213.
These methods include that separating, operate and express coding exempts from from least one heavy chain or all or part of of light chainThe nucleotide sequence of epidemic disease immunoglobulin variable domain.Described nucleic acid can be from producing the SDAB molecule for predetermined targetHybridoma obtains, and as described above, and obtains from other sources.It is then possible to by the restructuring of encoding humanized SDAB moleculeDNA clone is in suitable expression vector.
In certain embodiments, humanized SDAB molecule is taken by the conservative replacement of introducing, consensus sequence replacement, germlineGeneration and/or back mutation and optimised.The immunoglobulin molecules of described change can be by several technology known in the artIn any one prepare (such as, Teng et al., Proc.Natl.Acad.Sci.U.S.A. (NAS's journal),80:7308-7312 (1983)；Kozbor et al., Immunology Today (Immunol Today), 4:7279 (1983)；Olsson et al., Meth.Enzymol. (Enzymology method), 92:3-16 (1982)), and can be according to WO92/06193 or EPPrepared by the technology of 0239400.Technology for humanization SDAB molecule also provides in WO 06/122786
It is thin that SDAB molecule can also delete people T by the method specificity disclosed in WO 98/52976 and WO 00/34317Born of the same parents' epi-position or " going immunization " and be modified.In short, about the peptide being combined with II class MHC, the weight of SDAB molecule can be analyzedChain variable domains；These peptides represent potential t cell epitope (as defined in WO 98/52976 and WO 00/34317).ForThe t cell epitope that detection is potential, can apply referred to as the microcomputer modelling side of " peptide threading (peptide threading) "Method, and additionally can retrieve people II class MHC binding peptide data base and find motif present in VH and the VL sequence, as at WODescribed in 98/52976 and WO 00/34317.These motifs combine any one in 18 kinds of main II class MHC DR allotypes,Therefore potential t cell epitope is formed.Detected potential t cell epitope can be by replacing minority in variable domainsAmino acid residue or preferably replaced by single amino acids and be eliminated.Typically, conservative replacement is carried out.
Generally, but not exclusively, it is possible to use the aminoacid that the position in human germline antibody sequences is total.Such as,Human germ line sequences is disclosed in Tomlinson et al. .J.Mol.Biol. (J. Mol. BioL) 227:776-798 (1992)；Cook et al., Immunol.Today (Immunol Today) 16 (5): 237-242 (1995)；Chothia et al., J.Mol.Biol.(J. Mol. BioL) 227:799-817 (1992)；With Tomlinson et al., EMBO J.14:4628-4638 (1995)In.V BASE catalogue provides the panoramic catalogue of human normal immunoglobulin's variable region sequences (by Tomlinson, LA. et al. writing, MRCCentre for Protein Engineering (protein engineering MRC center), Cambridge, Britain).These sequences canFor use as the source of human sequence, such as, for framework region and CDRs.Can also use common somebody's framework region, such as, asDescribed in U.S.6,300,064.SDAB molecule can produce by being produced the host cell alive of this albumen by genetic modification.LosePass the raw albuminiferous method of engineered cells to be well known in the present art.(1990) are compiled for example, with reference to Ausabel et al.,Current Protocols in Molecular Biology (current molecular biological method) (Wiley, New York).SuchMethod includes coding and allows the nucleic acid of protein expression to be incorporated in host cell alive.These host cells can be to cultivate lifeLong bacterial cell, fungal cell or preferred zooblast.Bacterial host cell includes, but not limited to escherichia coli(Escherichia coli) cell.The example of suitable coli strain includes: HB101, DH5a, GM2929, JM109,KW251, NM538, NM539, and the arbitrary coli strain of foreign DNA can not be cut.The fungal host that can useCell includes, but not limited to saccharomyces cerevisiae (Sacchammyces cerevisiae), pichia pastoris phaff (PichiaAnd Eurotium (Aspergillus) cell pastoris).Some examples of the animal cell line that can use are CHO,VERO, BHK, HeLa, Cos, MDCK, 293,3T3 and WI38.The method of well known to a person skilled in the art can be used (such as, logicalCross conversion, virus infects and/or selects) set up new animal cell line.Optionally, albumen can be by host cell secretes to trainingSupport in base.
The SDAB molecule modified
The preparation of the present invention can include at least one such SDAB molecule, described SDAB molecule have framework region itThe ammonia that at least one amino acid position in one is different with the aminoacid sequence of naturally occurring domain (such as, VH domain)Base acid sequence.It should be understood that the aminoacid sequence of some SDAB molecules of the present invention (such as humanization SDAB molecule) can beAt least one amino acid position at least one framework region and naturally occurring domain (such as, naturally occurring VHI-I knotStructure territory) aminoacid sequence different.
Present invention additionally comprises the preparation of the derivant of SDAB molecule.Described derivant generally can be by modifying and specialIt not by chemistry and/or 5 biologys (such as, enzyme) modifying SDAB molecule and/or form the one of SDAB molecule disclosed hereinIndividual or multiple amino acid residue and obtain.Can repair by this way in the example of described modification, and SDAB molecular sequencesThe example of the amino acid residue (that is, on protein backbone, but preferably on side chain) of decorations, may be used for introducing such modificationMethod and technology and the potential purposes of such modification and advantage are clearly for technical staff.
Such as, such modification can include in SDAB molecule or on it introduce (such as, by covalently bound or with appointWhat his suitable mode) one or more functional groups, residue or part, and particularly give described SDAB molecule a kind of orThe characteristic of multiple needs or functional one or more functional group, residue or part.The example of described functional group is technology peopleMember institute is clearly.
Such as, described modification can include that introducing (such as, by covalent bond or in any other suitable) increasesAdd the half-life of SDAB molecule, dissolubility and/or absorption, immunogenicity and/or the toxicity of reduction SDAB molecule, eliminate or alleviateAny unwanted side effect of SDAB molecule and/or give other favourable characteristics of SDAB molecule and/or reduce unwantedOne or more functional groups of characteristic；Or two or more combination in any in aforementioned.The example of described functional group and introducingThe example of the technology of described functional group be technical staff institute clearly, and may be generally comprised in above-cited general backgroundIn all functional groups of mentioning and technology and the modification for pharmaceutical protein be known per se functional group and technology, particularlyFor modified antibodies or the functional group of antibody fragment (including ScFvs and single domain antibody) and technology, such as, for its referenceRemington ' s Pharmaceutical Sciences (Lei Mingdun pharmacopeia), the 16th edition, Mack Publishing Co.,Easton, PA (1980).Such as, described functional group to can be directly connected to (such as, being covalently attached to) of the present inventionOn SDAB molecule, or connect optionally by suitable joint or spacer, this be also technical staff institute clearly.About increasingThe immunogenic a kind of wide variety of technology adding half-life and/or minimizing pharmaceutical protein includes connecting suitable medicinal polymerizationThing such as PEG (PEG) or derivatives thereof (such as methoxyl group PEG or mPEG).Usually, can apply appointThe PEGization effect of what appropriate format, (includes but not limited to that (singly) domain resists for antibody and antibody fragment in such as this areaBody and ScFvs) PEGization effect；Such as, with reference to Chapman, Nat.Biotechnol. (Nature Biotechnol), 54:531-545(2002)；Veronese and Harris, Adv.Drug Deliv.Rev. (advanced drug delivery summary) 54:453-456(2003), Harris and Chess, Nat.Rev.Drug.Discov. (natural drug find summary), 2, (2003) and at WOIn 04/060965.Various reagent for albumen PEGization can also be purchased, such as from the Nektar Therapeutics of the U.S.Buy.
Preferably, application orientation PEGization, (see for example Yang etc., Protein especially by cysteine-residueEngineering (protein engineering), 16 (10): 761-770 (2003)).Such as, for the purpose of it, PEG may be coupled toIn SDAB molecule on naturally occurring cysteine residues, SDAB molecule can be modified, to be appropriately introduced into one or manyThe individual cysteine residues for connecting PEG, or, can will include that one or more cysteine for PEG connection is residualThe aminoacid sequence of base is fused to N-end and/or the C-end of the SDAB of the present invention, and all these uses are originally as art technologyProtein engineering known to personnel.
Preferably, about SDAB molecule, use molecular weight more than 5000, all such as larger than 10,000 and less than 200,000,All such as less than 100, the PEG of 000: such as, the PEG 20,000-80, in the range of 000.
About PEGization, it should be noted that generally present invention additionally comprises at one or more amino acid positions by PEGizationAny SDAB molecule, the most by this way, i.e. described PEGization (1) increase Half-life in vivo；(2) immunogen is reducedProperty；(3) provide about PEGization one or more other more favourable characteristics known per se；(4) described in having no substantial effect onThe affinity of SDAB molecule (such as, when by suitable detection assay, those detections described in the most following embodiment,Do not reduce the described affinity more than 90%, be preferably without reducing the described affinity more than 50%, and more preferablyDo not reduce the described affinity more than 10%)；And/or (4) do not affect any other spy needed of described SDAB moleculeProperty.Technical staff is it should also be apparent that suitable PEG-group and the method that specifically or non-specifically connects them.
Such as, the SDABs of PEGization is disclosed in the U.S. Provisional Application No. 61/265 submitted on July 16th, 2011, in 307,It is incorporated herein by reference.
In some embodiments, the SDAB of PEGization includes the SDAB molecule of the modification being connected with PEG polymer, whereinDescribed PEG polymer molecule is the side chain PEG polymer molecule of the group selecting free style (a)-(h) to form:
In some embodiments, the SDAB of described PEGization includes:
I one or more single antigen-binding domains that () is combined with one or more targets；
(ii) non-peptide linker；With
(iii) one or more polymer molecules,
Wherein said non-peptide linker is the part of formula (I):
Wherein
W1 and W2 is separately selected from key or NR1；
Y is key, with Ra or pyrrolidine-2, the 5-diketone substituted C1-4 alkylidene of 0-2 existence；
X is O, key or do not exist；
Z is O, NR3, S or key；
R1 and R3 is separately hydrogen or C1-6 alkyl；
R2 does not exists or one or more polymer moieties；
Ra is selected from hydroxyl, C1-4 alkyl or C1-4 alkoxyl；
M is 0 or 1；
N is 0,1,2 or 3；
P is 0,1,2,3 or 4.
In further embodiment, the SDAB of described PEGization is connected on PEG by the joint shown in following formula:
In particular embodiments, the SDAB molecule of described modification includes following structure:
It addition, modification the most less preferably includes the glycosylation that N-connects or O-connects, depend on for expressing describedThe host cell of SDAB molecule, this is usually used as common translation and/or a part for post translational modification.
The method preparing SDABs
SDAB molecule can produce by being produced the host cell alive of this albumen by genetic modification.Genetically modified cell is rawAlbuminiferous method is well known in the present art.(1990) are compiled, Current Protocols for example, with reference to Ausabel et al.In Molecular Biology (current molecular biological method) (Wiley, New York).Such method includes coding and permitsThe nucleic acid being permitted protein expression is incorporated in host cell alive.These host cells can be to cultivate the bacterial cell of growth, fungusCell or zooblast.Bacterial host cell includes, but not limited to Bacillus coli cells.The reality of suitable coli strainExample includes: HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and can not cut the arbitrary of foreign DNAColi strain.The fungal host cells that can use includes, but not limited to saccharomyces cerevisiae, pichia pastoris phaff and songMould Pseudomonas cell.Some examples of the animal cell line that can use are CHO, VERO, BHK, HeLa, Cos, MDCK, 293,3T3And WI38.The method of well known to a person skilled in the art (such as, infected by conversion, virus and/or select) can be used to set upNew animal cell line.Optionally, albumen can be by host cell secretes to culture medium.
In some embodiments, described SDAB molecule can produce in bacterial cell (such as, Bacillus coli cells).Such as, if SDAB is to be included suppressing biting of termination codon by between displaying entity and phage protein (or its fragment)Sequential coding in phage display vector, then can transfer to suppress the bacterial cell of termination codon by this vector nucleic acidIn.In this case, SDAB does not merges with gene III protein, and is secreted in pericentral siphon and/or culture medium.
SDAB molecule can also produce in eukaryotic cell.In one embodiment, SDAB molecule is in yeast cellsExpressing, described yeast cells such as Pichia sp. (Pichia) (see, e.g., Powers et al., J Immunol Methods.(J. Immunol. Methods) 251:123-35 (2001)), Hanseula or Sacchammyces.
In one embodiment, SDAB molecule produces in mammalian cell.For expression cloning antibody or its resistThe typical mammalian host cell of body-binding fragment includes that Chinese hamster ovary cell (Chinese hamster ovary celI) (includes dhfr-CHOCell, its description is at Urlaub and Chasin, Proc.Natl.Acad.Sci.USA (NAS's journal) 77:In 4216-4220 (1980), use DHFR selected marker, such as, as at Kaufman and Sharp, Mol.Biol. (molecular biosciencesLearn) described in 159:601-621 (1982)), lymphocyte series, such as, NS0 myeloma cell and SP2 cell, COS cell withAnd the cell from transgenic animal (such as, transgene mammal).Such as, described cell is breast epithelial cell.
In addition to the nucleotide sequence of coding SDAB molecule, recombinant expression carrier can also carry other sequence, such asThe sequence (such as, origin of replication) of regulation carrier duplication in host cell and selectable marker gene.Selectable marker gene promoteesEnter the selection to the host cell wherein having been incorporated into carrier (for example, with reference to U.S. Patent number 4,399,216；4,634,665；With 5,179,017).Such as, typically, selectable marker gene gives the drug resistance of the host cell having been incorporated into carrier, allSuch as G418, hygromycin or methotrexate resistance.
In the example system of recombinant expressed SDAB molecule, the recombinant expression carrier of coding single domain antibody chain passes throughThe transfection of calcium phosphate mediation is incorporated in dhfr-CHO cell.In recombinant expression carrier, antibody gene respectively operably withEnhancers/promoters regulating element (such as, derives from SV40, CMV, adenovirus etc., such as cmv enhancer/AdMLP promoterRegulating element or SV40 enhancer/AdMLP modulator promoter element) connect, thus drive this high-level gene to transcribe.Restructuring tableReaching carrier and also carry DHFR gene, it allows to utilize methotrexate to select/expand to select the CHO having transfected this carrier thinBorn of the same parents.The expression with permission antibody chain of the selected transformant host cell can be cultivated, and reclaim complete from culture mediumSingle domain.The Protocols in Molecular Biology of standard can be used to prepare recombinant expression carrier, and transfection host cell, selection convertsBody, cultivates host cell and reclaims antibody molecule from culture medium.Such as, some SDAB molecules can be divided by affinity chromatographFrom.
In one embodiment, described SDAB molecule such as purification described in WO 10/056550.Exemplary at oneEmbodiment in, by following, SDAB is come with one or more pollutant purification: make SDAB with one or more polluteThe mixture of thing is allowing SDAB to be attached to described holder with holder based on a-protein and/or ion exchange holderAbove or contact under conditions of being adsorbed onto on described holder；By washing under conditions of keeping being combined on described holder at SDABWash combined holder and remove one or more pollutant, and divided by the SDAB adsorbed with elution buffer elutingSon and from described holder selective elution SDAB.
SDAB molecule can also be produced by transgenic animal.Such as, U.S. Patent number 5,849,992 describe a kind of turningThe method expressing antibody in the mammary gland of gene mammal.Building transgenic, it includes breast specificity promoter and encoding antibodyThe nucleic acid of molecule and the signal sequence for secretion.Secretion is included by the milk of the female generation in described transgenic animalPurpose single domain in milk.Antibody molecule can from milk purification, or for some apply, can directly makeWith.
Preparation
The SDABs of the present invention can be configured to any pharmaceutical formulation.Described preparation can be liquid or dry product.Described preparationCan by mixing, be dried, lyophilizing, vacuum drying or the method for arbitrarily known preparation Pharmaceutical composition and produce.
Pharmaceutical formulation can be configured to the orderly of solution, microemulsion, dispersion liquid, liposome or other applicable high protein concentrationsStructure.Aseptic parenteral solution can be by following preparation: be combined in suitable solvent by the reagent as herein described of requirement, needsWhen wanting, there is a kind of composition listed above or the combination of Multiple components, then filtration sterilization.Generally, by by described hereinReagent be combined in sterile excipient and prepare dispersion liquid, described excipient includes basic disperse medium and arranges from belowOther the required compositions of those lifted.The appropriate mobility of solution can be maintained, such as, by using coating such as ovumPhospholipid, in the situation of dispersion liquid by maintain required for granularity and by use surfactant maintain.
By including the reagent postponing to absorb, such as, Monostearate and gelatin in the composition, injection group can be causedThe absorption of the prolongation of compound.
The preparation of SDAB molecule include SDAB molecule, can be as the compound of cryoprotective agent and buffer agent.Described systemThe pH of agent is usually pH 5.5-7.0, preferably from about pH 6.In some embodiments, preparation stores as liquid.Real at otherExecuting in scheme, preparation is prepared as liquid, is dried the most before storing, such as, by lyophilization or spray drying.It is driedPreparation can use as mummification compound, such as, as aerosol or powder, or reconstruct is to its initial concentration or another kind of denseDegree, such as, with water, buffer agent or other suitable liquid reconstruct.
Design SDAB molecular purifications method, the most cryodesiccated suitable to allow SDAB molecule to transfer to as frozen liqIn the preparation (such as, using histidine/sucrose preparation) of longer-term storage.Described preparation freezes together with the albumen of certain concentrationDry.Then, when needed, with suitable diluent (such as, water), the preparation of this lyophilizing is reconstructed, thus by original formulation compositionAgain the concentration needed it is dissolved to, it is common that identical with the concentration before lyophilizing or higher than the concentration before lyophilizing.
The preparation of lyophilizing can reconstruct, and depends on joining in lyophilized products relative to initial cryodesiccated liquid volumeWater or the amount of diluent, produce the preparation with the concentration different with initial concentration (that is, concentration before lyophilizing).By measuring antibodyOne or more parameters of integrity can identify suitable preparation.
The SDABs of the present invention can be as prepared described in WO 10/077422.The SDABs of the present invention can be by followingIllustrative methods is prepared: by 10 mixture lyophilizing of SDAB, cryoprotective agent, surfactant and buffer agent；And in dilutionAgent reconstructs the mixture of described lyophilizing, thus prepares described preparation.In particular embodiments, the SDABs of the present invention leads toCross the preparation of following method: in bacterial cultures, express SDAB；By chromatography purification step and/or ultrafiltration/dialysis step purificationSDAB；Including that concentration is about the sucrose of 5-10%, concentration is about the polysorbate80 of 0.01-0.02% and concentration is aboutThe histidine of 10-20mM or concentration are about in the preparation of Tris buffer (so that the pH of described preparation is about 5-7.5) of 20mMAdjust the concentration of SDAB to about 10-250mg/ml.
Therapeutic Method
SDAB molecule can individually or be subject to the second reagent (such as, the second treatment or pharmaceutically active agent) combined administrationExamination person (such as, people experimenter) treats or prevents (such as, alleviating or improve one or more associated symptoms) TNFaAssociated conditions, such as, inflammatory or autoimmune disorder.
The SDAB molecule of the present invention, such as includes two kinds of Anti-tumor necrosin (TNFa) SDABs and anti-human seralbuminThe polypeptide of albumen (HSA) SDAB, individually or can have this to need with the second agent combination as herein described for treatment or preventionImmune disorders in the people wanted.
Term " is treated " and is referred to be effectively improved the patient's condition, symptom or the parameter relevant to disease or prevent advancing to of diseaseThe degree of statistically significant or amount, mode and/or pattern to the detectable degree of those skilled in the art implement treatment.ControllingTreating in the situation of application, treatment can improve, cures, the disease that maintains in experimenter or the patient's condition or shorten disease or the patient's condition existsPersistent period in experimenter.In treatment use, experimenter is likely to be of the performance partially or completely of symptom.TypicallyIn situation, treatment improves the disease of experimenter or the patient's condition to the detectable degree of doctor, or prevents disease or the patient's condition from deteriorating.HaveAmount, mode or the pattern of effect can be different because of experimenter, and can be experimenter's customization.
When used herein, term " experimenter " and " patient " are used interchangeably.When used herein, " one is subject to termExamination person " and " multiple experimenter " refer to animal, such as, mammal, including non-primate (such as, cattle, pig, horse, donkey,Goat, camel, cat, Canis familiaris L., Cavia porcellus, rat, mice, sheep) and primate (such as, monkey, such as machin, gorilla,Chimpanzee and people).
The limiting examples of treatable immune disorders includes, but not limited to autoimmune disorder, such as, and jointInflammation (includes rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-relevantArthritis (lupus-associated arthritis) or ankylosing spondylitis), scleroderma, systemic lupus erythematosus (sle)(systemic lupus erythematosis), Sjogren syndrome, vasculitis, multiple sclerosis, autoimmunity first shapeAdenitis (autoimmune thyroiditis), dermatitis (dermatitis) (includes atopic dermatitis (atopicDermatitis) and eczematoid dermatitis (eczenatous dermatitis)), myasthenia gravis, inflammatory bowel (IBD), CrowGrace is sick, colitis (colitis), diabetes (diabetes mellitus) (I type)；Inflammatory disease, such as, the inflammatory of skinDisease (such as, psoriasis)；Acute inflammatory disorders (such as, endotoxemia (endotoxemia), sepsis (sepsis) andSepticemia (septicemia), toxic shock syndrome (toxic shock syndrome) and infectious disease (infectiousdisease))；Transplant rejection (transplant rejection) and allergy (allergy).In one embodiment,Described TNFa associated conditions is arthrtic condition, such as, and the disease of one or more in following: rheumatoid jointInflammation, juvenile rheumatoid arthritis (RA) (such as, moderate is to seriousness rheumatoid arthritis), osteoarthritis, psoriasisArthritis or ankylosing spondylitis, multi-joint Juvenile idiopathic arthritis (polyarticular juvenileIdiopathic arthritis, JIA)；Or psoriasis, ulcerative colitis, Crohn disease, inflammatory bowel and/or multipleHardening.
In certain embodiments, described SDAB molecule (or preparation) is used with the second therapeutic combination or is executed for combinationWith.Such as, for TNF-SDABs, the second reagent can be anti-TNF antibody or its combine the fragment of TNF, wherein the 2nd TNF resistsBody has the epitope specificity different from the SDAB molecule of the combination TNF in preparation.Can be with the SDAB co-formulation combining TNFOther nonrestrictive examples of reagent include, such as, cytokine inhibitor, growth factor receptor inhibitors, immunosuppressant,Antiinflammatory, metabolic poison, enzyme inhibitor, cytotoxic agent and cytostatic agent.In one embodiment, additionallyReagent is for arthritic standard care, includes, but not limited to non-steroidal anti-inflammatory agent (NSAIDs)；Corticosteroid, includingPrednisolone (prednisolone), prednisone (prednisone), cortisone (cortisone), and triamcinolone(triamcinolone)；With the antirheumatic (DMARDs) palliated a disease, such as methotrexate, oxychloroquine(hydroxychloroquine) (Plaquenil) and sulfur nitrogen sulphur pyridine (sulfasalazine), leflunomide(leflunomide)Tumor necrosis factor inhibitors, including Yi Naxipu (etanercept)Infliximab (infliximab)(with or without methotrexate), and adalimumab (adalimumab)Anti-CD 20 antibodies is (such as,), Soluble IL-1RI gene, such as Antril (Synergen)(asanakinra) (Kineret), gold, minocycline (minocycline)Penicillamine(penicillamine), and cytotoxic reagent, including azathioprine (azathioprine), cyclophosphamideAnd Cyclosporin A (cyclosporine) (cyclophosphamide).Described combined therapy can be advantageously employed relatively low-doseThe therapeutic agent used, therefore avoids the possible toxicity relevant to various single therapies or complication.
When other reagent is methotrexate, the dosage of methotrexate can be in the scope of the most about 7.5 to about 25mgIn.
SDAB molecule can be used or for executing with this with the form of liquid solution (such as, injection solution and infusion solution)With.Described compositions can be passed through parenteral pattern (such as, subcutaneous, intraperitoneal or intramuscular injection) or be used by suction.ShortLanguage " parenteral administration " and " passing through parenteral administration " are with being here and hereinafter meant that the mode of administration in addition to intestinal and local application, logicalBe often to be used by injection, and include subcutaneously or intramuscularly using, and intravenous, capsule in, ophthalmic, intracardiac, Intradermal, peritoneumIn, under trachea, epidermis, under capsule, under arachnoidea, in spinal column, epidural and breastbone inner injection and infusion.An embodimentIn, preparation as herein described passes through subcutaneous administration.
The present inventor estimates the ozoralizumab bispecific interaction sites with TNF α for treatment immune disordersIt is useful especially.Therefore, the method that the present invention relates to treat immune disorders in this people needed, described method include toThis people uses the polypeptide of the polypeptide competition with the aminoacid sequence including SEQ ID NO:1 (ozoralizumab).
Cheat regimen
Ozoralizumab be spaced between twice administration at least surrounding subcutaneous administration time demonstrate treatment rheumatoid closeEffect that joint is scorching.By contrast, aboutThe dosage regimen recommended is to use every two weeks, andMust use by intravenous.Accordingly, with respect to prior art situation, the dosage regimen of the present invention provides the advantage that.
The present invention SDAB molecule with the dosage of every 30mg and 80mg used for 4 weeks demonstrated treatment disease meritEffect.Modelings based on these efficacy outcomes show with higher dosage (such as 120-200mg or 200-400mg), the present invention'sSDAB molecule is effective to treatment disease at every 6 or 8 weeks or time February uses.This useful pattern causes the injection burden reduced.
It addition, modeling also shows that the severe infections (SI) that ozoralizumab treats is less than Embrel and Infliximab listAnti-severe infections.Especially, compared with anti-TNF a inhibitor of the prior art, it is right that the SDAB molecule of the present invention demonstratesThe very favorable benefit (effect) of dangerous (SI effect) ratio.
Therefore, the SDAB molecule of the present invention can be used with the dosage in the range of 30-200mg for every 4,6 or 8 weeks.Have especiallyThe dosage of effect is 30-400mg.In particular embodiments, described dosage includes about 5,10,15,20,25,30,80,100,120,140,160,180,200,225,250,275,300,320,350,375 or 400mg SDAB molecules.
The single dose of about 30-200mg or even 30-400mg can about every day, every other day, biweekly, every 1,2,3,4,5,6,7,8,9 or 10 weeks or every 1 or use February.
In particular embodiments, about 30,80,120,160,200,240,280,320,360 or 400mg include SEQIt is tested that the dosage of the polypeptide of the aminoacid sequence of ID NO:1 (ozoralizumab) is administered to people every about 4,6 or 8 weeks or FebruaryPerson.In some embodiments, described experimenter suffers from rheumatoid arthritis.
In further embodiment, the dosage of described SDAB molecule is about 1, and 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395 or 400mg.
In some embodiments, described dosage is about 3.6, and 10.8,36,72,144 or 288mg.
In particular embodiments, described SDAB molecule is PEGization, and dosage is about 3.6,10.8,36,72,144 or 288mg.
For using to adolescent patient, dosage should be adjusted according to the body weight of described patient.In concrete realityExecuting in scheme, described dosage is about 0.1, and 0.38,1,2,3,3.5,4,4.5 or 5mg/kg.
Experimenter can use methotrexate therapy simultaneously.In some embodiments, described experimenter is starting administrationMethotrexate has been accepted at least about 6 or 12 weeks before ozoralizumab.Methotrexate can be by the most suitable approachUse, and dosage can be in the range of the most about 7.5-25mg.
The method determining effect
Effect of any concrete SDAB molecule or dosage regimen can by those skilled in the art can method trueFixed.In short, during clinical trial, patient can be watched by medical personnel, and combined by arbitrary standards and assessDisease condition.The improvement of patient disease situation is determined based on these standards at multiple time points, and by these determined combinationsDraw to assess the effect treated for PATIENT POPULATION.
In exemplary embodiment, any mark that the progression of disease of rheumatoid arthritis can be listed by table 2Accurate or all standards are measured.
Table 2