CN107501412A - Saltant type bispecific antibody and its application - Google Patents

Abstract

The present invention relates to a kind of saltant type bispecific antibody, antibody structure includes anti CD20VL mutant, the first connection peptide, anti CD20VH mutant, the second connection peptide, anti CD3VH mutant, the 3rd connection peptide and the anti CD3VL mutant being sequentially connected.The structure of the saltant type bispecific antibody is simple, and tissue penetration capacity is stronger.And the saltant type bispecific antibody can rely on rAAV directly in body oneself expression, have the advantages that accurate target spot, secure persistent, high specific, evident in efficacy, price can be born, have wide practical use in antineoplastic.

Description

Saltant type bispecific antibody and its application

Technical field

The present invention relates to biological technical field, more particularly to a kind of saltant type bispecific antibody and its application.

Background technology

Lymthoma is initiated by the malignant tumour of lymphohematological, is mainly shown as Silent Neuritis enlargement of lymph nodes, liver and spleenEnlargement, each histoorgan of whole body can be involved, and the heat that occurs together, night sweat, become thin, the constitutional symptom such as itch.Lymphomatosis is because unclear, and oneAs think may with gene mutation and virus and other pathogenic infections, radioactive ray, chemicals, merge autoimmunity disease etc.It is relevant.

B cell lymphoma is a kind of most common lymthoma, accounts for the 50%~60% of lymthoma.The disease is at me in recent yearsThe incidence of disease of state quickly raises, and seriously endangers the health of our people.One line therapeutic scheme is Rituximab at present(Roche) and CHOP therapeutic alliances (i.e. R-CHOP schemes), although the patient for having more than 50% can be after this therapeutic scheme be receivedIt is cured, but it is expensive, and medical expense is up to hundreds thousand of members, and China patient is difficult to bear.There is an urgent need to a kind of new for they, the B cell lymphoma therapeutic agent that evident in efficacy and price can be born.

The development of nearly 30 years is passed through in gene therapy, is expanded to via the treatment for being used primarily for monogenic inheritance diseaseMore than 6000 kinds of disease, including cancer, AIDS, hepatitis B, autoimmune disease and metabolic disease etc..Holland in 2012The Glybera of UniQure companies goes through to list as the first gene therapy medicament in the whole world, formally opens the new of gene therapyMileage.

Bispecific antibody refers to the artificial antibody containing two species-specific antigen binding sites, is to get most of the attention in recent yearsClinical practice novel antibodies.It is broadly divided into 2 classes:Bispecific antibody containing Fc areas and the bispecific without Fc areas resistBody.The stability of antibody can be ensured comprising Fc areas, maintains the drug effect concentration of long period in vivo, but produces purification difficult, pointSon amount is big, tissue permeability is undesirable.Bispecific antibody production purifying process not comprising Fc areas is relatively easy, and because of its pointSon amount is small, is more easy to penetrate into function is played in histocyte, but its half-life short is a big inferior position.

The content of the invention

Based on this, it is necessary to provide one kind can be easy to production, easily penetrate into tumor tissues and can and antigen with compared withThe saltant type bispecific antibody of strong binding ability and its application.

A kind of saltant type bispecific antibody, including be sequentially connected anti-CD20VL mutant, first connection peptide,Anti-CD20VH mutant, the second connection peptide, anti-CD3VH mutant, the 3rd connection peptide and anti-CD3VL mutant；

Wherein, the anti-CD20VL mutant is more including being made up of the amino acid sequence shown in SEQ ID No.1Peptide, the anti-CD20VH mutant include the polypeptide being made up of the amino acid sequence shown in SEQ ID No.2, the anti-CD3VH mutant includes the polypeptide being made up of the amino acid sequence shown in SEQ ID No.3, the anti-CD3VL mutant bagThe polypeptide being made up of the amino acid sequence shown in SEQ ID No.4 is included, the saltant type bispecific antibody is single-chain antibody.

The saltant type bispecific antibody (BsAb) include be sequentially connected anti-CD20VL mutant, first connection peptide,Anti-CD20VH mutant, the second connection peptide, anti-CD3VH mutant, the 3rd connection peptide and anti-CD3VL mutant.Wherein, anti-CD20VL mutant and anti-CD20VH mutant are respectively by VL (light chain) area of anti-CD-20 monoclonal antibodyDomain and VH (heavy chain) region carry out partial amino-acid and are mutated to obtain.Anti-CD3VH mutant and anti-CD3VL mutant differencePartial amino-acid is carried out by VH (heavy chain) regions of CD 3-resisting monoclonal antibody and VL (light chain) region to be mutated to obtain.The saltant typeThe antibody regions of bispecific antibody are formed by the VL regions and VH regions of CD20 antibody and CD3 antibody, simple in construction, are penetrated intoAbility in tumor tissues is stronger.And after anti-CD20VL and VH function region mutations, the anti-CD20VL mutant of acquisition andAnti-CD20VH mutant can fold the antibody variable region knot for forming specific recognition CD20 molecules with the first connection peptide connectionStructure.After anti-CD3VL and VH function region mutations, the anti-CD3VH mutant and anti-CD3VL mutant of acquisition are with the 3rdPeptide connection is connected, the antibody variable plot structure for forming specific recognition CD3 molecules can be folded.Whole saltant type bispecific antibodyEach functional area is in turn connected to form single-stranded shape, and N-terminal specific recognition CD20 is formed by covalent bond equimolecular intermolecular forcesTwo complete antibody variable region structures of molecule, C-terminal specific recognition CD3 molecules are stronger with the binding ability of corresponding antigen.

In one embodiment, the saltant type bispecific antibody also includes Flag label proteins and His label eggsIn vain, the Flag label proteins mark the N-terminal in the anti-CD20VL mutant, and the His labels egg mark is describedThe C-terminal of anti-CD3VL mutant.

In one embodiment, the saltant type bispecific antibody includes:(a) as the ammonia shown in SEQ ID No.5The polypeptide of base acid sequence composition, it is (b), same with least 98% with the polypeptide of the amino acid sequence composition shown in SEQ ID No.5Source property and with the saltant type bispecific antibody activity polypeptide, or (c), as the amino acid sequence shown in SEQ ID No.5The polypeptide of composition is arranged, wherein one or more amino acid, which are lacked, substituted or increased, to be obtained and have the saltant type double specialThe polypeptide of property antibody activity.

In one embodiment, the saltant type bispecific antibody includes：(a), as the core shown in SEQ ID No.6What the nucleotide sequence shown in the polypeptide that the polynucleotide encoding of nucleotide sequence composition obtains, (b) and SEQ ID No.6 formedPolynucleotide encoding of the polynucleotides with least 98% homology obtains and with saltant type bispecific antibody activityPolypeptide, or (c), the polynucleotides that are made up of the nucleotide sequence shown in SEQ ID No.6, wherein one or more base quiltsMissing, substitute or increase the polypeptide that obtained polynucleotide encoding obtains and has saltant type bispecific antibody activity.

A kind of saltant type bispecific antibody expressing gene, for encoding mutant type bispecific antibody, the saltant typeBispecific antibody include be sequentially connected anti-CD20VL mutant, first connection peptide, anti-CD20VH mutant, secondConnect peptide, anti-CD3VH mutant, the 3rd connection peptide and anti-CD3VL mutant；

Wherein, the anti-CD20VL mutant is more including being made up of the amino acid sequence shown in SEQ ID No.1Peptide, the anti-CD20VH mutant include the polypeptide being made up of the amino acid sequence shown in SEQ ID No.2, the anti-CD3VH mutant includes the polypeptide being made up of the amino acid sequence shown in SEQ ID No.3, the anti-CD3VL mutant bagThe polypeptide being made up of the amino acid sequence shown in SEQ ID No.4 is included, the saltant type bispecific antibody is single-chain antibody.

In one embodiment, the saltant type bispecific antibody expressing gene includes：(a), SEQ ID No.6 institutesNucleotide sequence shown in the nucleotide sequence shown, (b) and SEQ ID No.6 has the nucleotides sequence of at least 98% homologyRow, or the nucleotide sequence shown in (c), SEQ ID No.6, wherein one or more bases are lacked, substituted or increased and obtainedNucleotide sequence.

A kind of recombinant expression carrier, there is the saltant type bispecific antibody expressing gene described in any of the above-described.

A kind of host cell, there is the saltant type bispecific antibody expressing gene described in any of the above-described.

A kind of gene therapeutic agents, including adeno-associated virus and carry any of the above-described institute in the adeno-associated virusThe saltant type bispecific antibody expressing gene stated.

Saltant type bispecific antibody described in any of the above-described or the saltant type bispecific antibody described in any of the above-describedApplication of the expressing gene in anti-tumor drug is prepared.

Brief description of the drawings

Fig. 1 is the structural representation of an embodiment saltant type bispecific antibody；

Amino acid of the anti-CD20VL regions with anti-CD20VL mutant that Fig. 2 is Obinutuzumab (GA101)Sequence alignment figure；

Amino acid of the anti-CD20VH regions with anti-CD20VH mutant that Fig. 3 is Obinutuzumab (GA101)Sequence alignment figure；

Amino acid sequence of the anti-CD3VH regions with anti-CD3VH mutant that Fig. 4 is Blinatumomab (OKT3)Comparison chart；

Amino acid sequence of the anti-CD3VL regions with anti-CD3VL mutant that Fig. 5 is Blinatumomab (OKT3)Comparison chart；

Fig. 6 is the collection of illustrative plates of the plasmids of pSNAV 2.0 in embodiment 3；

Fig. 7 is the standard curve that the standard items plasmid QPCR amplifications of different copy gradients in test case one obtain；

Fig. 8 is the BsAb expressed in test case two after rAAV-BsAb viruses infection C2C12 cells through Western-Blotting analysis result figures；

Fig. 9 is the combination feelings of BsAb and effector cell and target cell under Flow cytometry different condition in test case threeCondition comparison chart；

Figure 10 is the streaming scatterplot result comparison chart for killing target cell in test case four under different condition.

Embodiment

In order to facilitate the understanding of the purposes, features and advantages of the present invention, with reference to specific embodiment andAccompanying drawing is described in detail to the embodiment of the present invention.Elaborate in the following description many details in order toFully understand the present invention.But the invention can be embodied in many other ways as described herein, art technologyPersonnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not by following public specific implementationLimitation.

Refer to Fig. 1, the saltant type bispecific antibody (BsAb) of an embodiment, including the anti-being sequentially connectedCD20VL mutant, the first connection peptide, anti-CD20VH mutant, the second connection peptide, anti-CD3VH mutant, the 3rd connectConnect peptide and anti-CD3VL mutant.Wherein, anti-CD20VL mutant is included as the amino acid shown in SEQ ID No.1The polypeptide of sequence composition, anti-CD20VH mutant include the polypeptide being made up of the amino acid sequence shown in SEQ ID No.2,Anti-CD3VH mutant includes the polypeptide being made up of the amino acid sequence shown in SEQ ID No.3, anti-CD3VL mutantIncluding the polypeptide being made up of the amino acid sequence shown in SEQ ID No.4.The saltant type bispecific antibody is single-chain antibody.

Specifically, anti-CD20VL mutant and anti-CD20VH mutant are respectively by anti-CD-20 monoclonal antibodyVL (light chain) regions and VH (heavy chain) region carry out partial amino-acid and are mutated to obtain.Anti-CD3VH mutant and anti-CD3VLMutant carries out partial amino-acid by VH (heavy chain) regions of CD 3-resisting monoclonal antibody and VL (light chain) region respectively and is mutatedArrive.

In present embodiment, anti-CD20VL mutant includes what is be made up of the amino acid sequence shown in SEQ ID No.1Polypeptide.Anti-CD20VL mutant is obtained by Obinutuzumab (GA101) anti-CD20VL region mutagenesis.Specifically,Obinutuzumab (GA101) anti-CD20VL regions by 116 Amino acid profiles, and anti-CD20VL mutant by109 Amino acid profiles.Obinutuzumab (GA101) anti-CD20VL regions and the amino of anti-CD20VL mutantSour comparison chart is as shown in Figure 2.Following mutation is passed through in Obinutuzumab (GA101) anti-CD20VL regions：D1Q, M4L,L (leucine) is lacked at T5S, T7S, No. 9 positions, S10A, S11I, P13S, V14A, T15S, P19K, A20V, S21T, I22M,S23T, S26A, K28S, 30~35 amino acids L, L, H, S, N are lacked, I36V, T37S, L39I, Y40H, Y42F, L43Q,Q48S, Q51K, L52P, L53W, Q56A, M57T, V61A, D66V, D76S, F77Y, T78S, K80T, V89A, G90A, V91T,A95Q, N97W, L98T, E99S, L100N, Y102P, V110L and the 115th T (threonine) and the 116th V (valine) lackLose.Wherein, first D (aspartic acid) is changed to Q (paddy ammonia in D1Q expression Obinutuzumab GA101 anti-CD20VLAcid amides), remaining is by that analogy.

In present embodiment, anti-CD20VH mutant includes what is be made up of the amino acid sequence shown in SEQ ID No.2Polypeptide.Anti-CD20VH mutant is obtained by Obinutuzumab (GA101) anti-CD20VH region mutagenesis.Specifically,Obinutuzumab (GA101) anti-CD20VH regions by 119 Amino acid profiles, and anti-CD20VH mutant by123 Amino acid profiles.Obinutuzumab (GA101) anti-CD20VH regions and the amino of anti-CD20VH mutantSour comparison chart is as shown in Figure 3.Following mutation is passed through in Obinutuzumab (GA101) anti-CD20VH regions：V5Q, S7P,V11L, K12V, S16A, V20M, T27Y, A28T, S30T, Y31S, S32Y, W33N, I34M, N35H, R38K, A40T, N43R,M48I, R50A, F52Y, D55N, D59S, G62Q, R67K, V68A, I70L, T76S, E82Q, R87T, T91S, T94Y, T95Y,Insertion two amino acid of D, W behind N99S, V100T, F101Y, D102Y, Y104G, 104 site, W105Y, L106F, V107N,Two amino acid of A, S are inserted after Y108V, Q111A, L114T, S119A and the 119th amino acids.Wherein, V5Q is representedThe 5th V (valine) is changed to Q (glutamine) in Obinutuzumab GA101 anti-CD20VH, and remaining is with suchPush away.

In present embodiment, anti-CD3VH mutant includes what is be made up of the amino acid sequence shown in SEQ ID No.3Polypeptide.Anti-CD3VH mutant is obtained by Blinatumomab (OKT3) anti-CD3VH region mutagenesis.Specifically,Blinatumomab (OKT3) anti-CD3VH regions are by 119 Amino acid profiles, and anti-CD3VH mutant is by 119 ammoniaBase acid is formed.Blinatumomab (OKT3) anti-CD3VH regions and the amino acid alignment figure of anti-CD3VH mutant are such asShown in Fig. 4.Following mutation is passed through in Blinatumomab (OKT3) anti-CD3VH regions：I2V, K3Q, Q5V, L11V, A12K,R13K, M20V, T24A, K38R, R40A, N61A, Q62D, K63S, F64V, D66G, K67R, A68F, L70T, S76T, Q82E,T87R, S91T and V93T.Second I is (different bright in wherein I2V expression Blinatumomab (OKT3) anti-CD3VH regionsPropylhomoserin) V (valine) is changed to, remaining is by that analogy.

In present embodiment, anti-CD3VL mutant includes what is be made up of the amino acid sequence shown in SEQ ID No.4Polypeptide.Anti-CD3VL mutant is obtained by Blinatumomab (OKT3) anti-CD3VL region mutagenesis.Specifically,Blinatumomab (OKT3) anti-CD3VL regions are by 106 Amino acid profiles, and anti-CD3VL mutant is by 106 ammoniaBase acid is formed.Blinatumomab (OKT3) anti-CD3VL regions and the amino acid alignment figure of anti-CD3VL mutant are such asShown in Fig. 5.Following mutation is passed through in Blinatumomab (OKT3) anti-CD3VL regions:Q3V, I10T, M11L, A13L,K18R, V19A, M21L, T22S, S27Q, S39P, T41K, S42A, Y59A, S69D, S75N, M77L, A99G, L103V andL105I.3rd Q glutamine in wherein Q3V expression Blinatumomab (OKT3) anti-CD3VL regions) it is changed to V(valine), remaining is by that analogy.

Specifically, saltant type bispecific antibody also includes Flag labels and His labels, and Flag labels are marked in anti-The N-terminal of CD20VL mutant, His labels mark the C-terminal in anti-CD3VL mutant.Because the saltant type of the present invention is double specialProperty antibody contain Flag label H is labels, therefore after the expression of saltant type bispecific antibody, when doing external functional verification, justIn with EUSA (ELISA) or other method measure cell culture fluid in saltant type bispecific antibody albumenConcentration, expressed saltant type bispecific antibody can also be purified with nickel post or immune-affinity chromatography, improve purity.

In present embodiment, saltant type bispecific antibody also includes signal peptide (signal peptide, SP), such asIgG signal peptides, IgG signal peptides are connected with Flag labels.

In present embodiment, saltant type bispecific antibody includes:(a) as the amino acid sequence shown in SEQ ID No.5The polypeptide of amino acid sequence composition shown in the polypeptide of composition, (b) and SEQ ID No.5 has at least 98% homology and toolHave a polypeptide of saltant type bispecific antibody activity, or (c), be made up of the amino acid sequence shown in SEQ ID No.5Polypeptide, wherein one or more amino acid, which are lacked, substituted or increased, to be obtained and is lived with the saltant type bispecific antibodyThe polypeptide of property.BsAb gene expressions are：IgG-SP-Flag-anti-CD20VL mutant-linker1-anti-- 6 × his of CD20VH mutant-linker2-anti-CD3VH mutant-linker3-anti-CD3VL mutant.

First connection peptide (linker1) connects the N-terminal of the C-terminal of anti-CD20VL mutant and anti-CD20VH mutantConnect, amino acid sequence is：GGGGSGGGGSGGGGS (SEQ ID No.5 the 137th~151).Second connection peptide(linker2) C-terminal of anti-CD20VH mutant is connected with the N-terminal of anti-CD3VH mutant, amino acid sequence is：GGGGS (SEQ ID No.5 the 275th~279).3rd connection peptide (linker3) by anti-CD3VH mutant C-terminal withAnti-CD3VL mutant N-terminal connects, and amino acid sequence is：GEGTSTGSGGSGGSGGAD (SEQ ID No.5 the 399th~416).Junction fragment is shorter, by each function and is sequentially connected the single-stranded saltant type bispecific antibody of composition.

Further, saltant type bispecific antibody includes：(a), it is made up of the nucleotide sequence shown in SEQ ID No.6The obtained polypeptide of polynucleotide encoding, (b), have with the polynucleotides of the nucleotide sequence composition shown in SEQ ID No.6The polynucleotide encoding of at least 98% homology obtains and has the polypeptide of saltant type bispecific antibody activity, or (c),The polynucleotides being made up of the nucleotide sequence shown in SEQ ID No.6, wherein one or more bases are lacked, substituted or increasedThe polynucleotide encoding for adding to obtain obtains and with the polypeptide of saltant type bispecific antibody activity.

Above-mentioned saltant type bispecific antibody at least has the advantages that：(1) antibody regions are dashed forward by anti-CD20VLVariant-linker1-anti-CD20VH mutant-linker2-anti-CD3VH mutant-linker3-anti-CD3VL mutant is formed, the variable region of variable region and anti-cd 3 antibodies from anti-CD 20 antibodies form a single-stranded CD20 ×CD3 bispecific antibodies, simple in construction, tissue permeability is strong.(2) anti-CD20 antibody and anti-CD3 antibody after being mutatedDisplay has stronger binding activity with corresponding CD20, CD3 molecule respectively.(3) CD20 antibody can target B cell lymphoma tableFace antigen, CD3 antibody can targeting T-cells surface antigen, import cell after, in environment existing for B lymphoma cells, CD20× CD3 bispecific antibodies can activate human T lymphocyte, and specific mediate T cell kills B lymphoma cells.(4) mutationType bispecific antibody can rely on rAAV and directly hold time longer in body oneself expression, blood concentration.

The saltant type bispecific antibody expressing gene of one embodiment, should for encoding mutant type bispecific antibodySaltant type bispecific antibody includes the anti-CD20VL mutant, the first connection peptide, anti-CD20VH mutation being sequentially connectedBody, the second connection peptide, anti-CD3VH mutant, the 3rd connection peptide and anti-CD3VL mutant.Wherein, anti-CD20VL mutant includes the polypeptide being made up of the amino acid sequence shown in SEQ ID No.1, and anti-CD20VH mutant includesThe polypeptide being made up of the amino acid sequence shown in SEQ ID No.2, anti-CD3VH mutant are included as shown in SEQ ID No.3Amino acid sequence composition polypeptide, anti-CD3VL mutant include be made up of the amino acid sequence shown in SEQ ID No.4Polypeptide, the saltant type bispecific antibody is single-chain antibody.

Specifically, the structure of saltant type bispecific antibody can be found in described above, and therefore not to repeat here.

In one embodiment, saltant type bispecific antibody expressing gene includes：(a), shown in SEQ ID No.6Nucleotide sequence shown in nucleotide sequence, (b) and SEQ ID No.6 has the nucleotide sequence of at least 98% homology, or(c), the nucleotide sequence shown in SEQ ID No.6, wherein one or more bases are lacked, substituted or increased obtained nucleosidesAcid sequence.

The saltant type bispecific antibody expressing gene, which can encode, obtains saltant type bispecific antibody, so as to be used for BThe treatment of cell lymphoma.

It is appreciated that polymorphism and variation due to expressing gene, coding identical albumen may have diversified formsExpressing gene.If base is lacked, substituted or increased in expressing gene, or the missing of amino acid, insertion, substitution or otherVariation, lacked, substituted or increased so as to cause the amino acid sequence of protein one or more amino acid occur.Expression obtainsProtein be different from corresponding protein, but be referred to as function with more peptide or proteins that the protein does not have obvious function differenceEquivalent variant.There is no the polypeptide or egg of obvious function difference with saltant type bispecific antibody therefore, it is possible to express to obtainWhite expressing gene will be understood that the expressing gene being equal with the saltant type bispecific antibody expressing gene in the application.With dashing forwardIt is double with the saltant type in the application that modification bispecific antibody does not have more peptide or proteins of obvious function difference all to will be understood thatThe equivalent albumen of specific antibody.

The recombinant expression carrier of one embodiment, there is above-mentioned saltant type bispecific antibody expressing gene.

Specifically, recombinant expression carrier is the pUC57 carriers for inserting the nucleotide sequence shown in SEQ ID No.6, shouldExpression vector can be used in cloning above-mentioned saltant type bispecific antibody sequence.

The host cell of one embodiment, there is above-mentioned saltant type bispecific antibody expressing gene.

Specifically, saltant type bispecific antibody expressing gene is entered in host cell by way of plasmid conversion, is led toPlasmid replication is crossed to be expanded.Host cell is, for example, competent escherichia coli cell etc..

The gene therapeutic agents of one embodiment, including the saltant type of adeno-associated virus and carrying in adeno-associated virus are doubleSpecific antibody expressing gene.The particular sequence of saltant type bispecific antibody expressing gene can be found in described above.

Saltant type bispecific antibody expressing gene is carried in adeno-associated virus, and B cell is targetted with adeno-associated virusLymthoma, and become " factory " that therapeutic agent produces in body, realize its " marketing one's own products " in body.So it can both protectDemonstrate,prove B cell lymphoma therapeutic effect, use cost can be greatly reduced again, will turn into a kind of target spot precisely, secure persistent, Gao TeThe gene therapeutic agents for the B cell lymphoma that different in nature, evident in efficacy, price can be born.

The expressing gene of above-mentioned saltant type bispecific antibody or above-mentioned saltant type bispecific antibody prepare it is antitumorMedicine in application.

In the application of an embodiment, anti-tumor drug is specially the medicine of anti-B cell lymphoma.Saltant type is double specialCD20 antibody on heterogenetic antibody can be directed to B cell lymphoma surface antigen, and CD3 antibody can be directed to t cell surface antigen,Cell after importing cell, saltant type bispecific antibody can target B cell lymphoma, and can activate human T lymphocyte,So as to kill B cell lymphoma, target spot is accurate, specificity is high.

Further, the anti-tumor drug is the medicine of anti-diffusivity B cell lymphoma.

Specifically, the anti-tumor drug is active component by above-mentioned saltant type bispecific antibody.Certainly, it is antitumorPharmaceutic adjuvant can also be added in medicine.The formulation of medicine can be the common formulations such as injection, powder-injection, lyophilized preparation.

Above-mentioned saltant type bispecific antibody or above-mentioned saltant type bispecific antibody expressing gene bleach what coding obtainedAntibody structure is simple, and the ability penetrated into tumor tissues is stronger.And after anti-CD20VL and VH function region mutations, acquisitionAnti-CD20VL mutant and anti-CD20VH mutant are connected with the first connection peptide, can be folded and be formed specific recognition CD20The antibody variable plot structure of molecule.After anti-CD3VL and VH function region mutations, the anti-CD3VH mutant and anti-of acquisitionCD3VL mutant can fold the antibody variable plot structure for forming specific recognition CD3 molecules with the 3rd connection peptide connection.EntirelyThe each functional area of saltant type bispecific antibody is in turn connected to form single-stranded shape, is formed by covalent bond equimolecular intermolecular forcesN-terminal specific recognition CD20 molecules, the complete antibody variable region structure of C-terminal specific recognition CD3 molecules and corresponding antigenBinding ability is stronger.And the saltant type bispecific antibody can rely on rAAV directly in body oneself expression, blood concentration dimensionIt is longer to hold the time.Anti-tumor drug can be used as, there is accurate target spot, secure persistent, high specific, evident in efficacy, priceThe advantages that can bearing, have wide practical use in antineoplastic.

It is specific embodiment part below.

In following examples, unless otherwise instructed, the experimental method of unreceipted actual conditions, generally according to normal condition,For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Mannies A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is realTest guide [M] (Beijing：Science Press, 1992) method that condition or kit manufacturer described in are recommended is realized.Reagent used in embodiment is commercially available.Wherein FastDigest EcoR I, FastDigest Bam HI endonucleasesEnzyme and Lipofectamine 2000 are purchased from U.S. Thermo.Glue reclaim kit, PCR Cleanup cleaning agents boxes and without interiorToxin plasmid extracts kit is purchased from Beijing Tiangeng.T4DNA ligases are purchased from NEB.AAV-293 cell deriveds in U.S. ATCC,DMEM culture mediums and 10% hyclone are purchased from Gibco companies of the U.S., and SYBR Green mix are purchased from the precious biology in Dalian.

Not specified, BsAb represents bispecific antibody.RAAV-BsAb viruses represent that carrying saltant type bispecific resistsThe adeno-associated virus of body expressing gene, gene therapeutic agents.

Embodiment 1 prepares saltant type bispecific antibody (BsAb) expressing gene

According to nucleotide sequence shown in SEQ ID No.6, EcoR I restriction enzyme site sequences are added at 5 ' ends, are added at 3 ' endsBam HI restriction enzyme site sequences, commission Suzhou Hong Xun bio tech ltd synthesize to the sequence.

Embodiment 2 saltant type bispecific antibody (BsAb) retrieval

By the BsAb gene integrations synthesized in embodiment 1 on pUC57 carriers, pUC57-BsAb is named as.

Endonuclease reaction system is prepared according to following system, digestion is carried out to pUC57-BsAb plasmids：

Component	Volume/content
		10×FastDigest Buffer	2μL
EcoR I endonucleases	1μL
		Bam HI endonucleases	1μL
pUC57-BsAb	2μg
		ddH2O	Complement to 20 μ L

Above-mentioned reaction system is placed in 37 DEG C of water-baths, reacts the laggard row agarose gel electrophoresis of 1h, and return by glueReceive purifying BsAb target gene fragments.

Embodiment 3 builds the recombinant vector of saltant type bispecific antibody (BsAb)

Prepare rAAV plasmids (being specially the plasmids of pSNAV 2.0), plasmid map is as shown in Figure 6.According to the side in embodiment 2Method, digestion processing is carried out to the plasmids of pSNAV 2.0 with EcoR I and Bam HI endonucleases, then using PCR CleanupCleaning agents box reclaims digestion products, the pSNAV2.0 plasmids linearized.

Coupled reaction system is prepared by following system, by the BsAb target gene fragment obtained in embodiment 2 and linearisationThe plasmids of pSNAV 2.0 connect that (wherein BsAb target gene fragment and the mol ratio of pSNAV2.0 plasmids of linearisation be3:1)：

Above-mentioned reaction system is placed in 16 DEG C of water-baths, reaction is overnight.Connection product transformed competence colibacillus bacillus coli DH 5α, it is applied on the LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C of overnight incubations, picking single bacterium colony.The single bacterium colony of picking is put(contain 100 μ g/mL ampicillins) in LB liquid medium, 37 DEG C, 200rpm shaken cultivations are stayed overnight, and bacterium solution then is delivered into Soviet UnionZhou Hongxun bio tech ltd is sequenced.It is required pSNAV-BsAb plasmids that correct plasmid, which is sequenced,.Expand trainingBring enterobacteria up, then extract pSNAV-BsAb plasmids with endotoxin-free plasmid extraction kit.

It is viral (gene therapeutic agents) that embodiment 4 prepares rAAV-BsAb

(1) recovery and culture of AAV-293 cells

A. prepare the water-bath that temperature is 37 DEG C, the cell frozen is taken out from liquid nitrogen container, be put into rapidly in water-bath and fastSpeed is rocked, and is completely dissolved cell solution in 1~2min as far as possible；

B. cell solution is transferred in 15mL centrifuge tubes, and complete medium fresh plus 9mL wherein, after mixingCentrifugation, 150 × g, 5min；

C. remove supernatant, add the fresh complete mediums of 1mL and softly dispel cell, be transferred to 10cm culture dishes, Mei GepeiFoster ware supplies 10mL culture mediums, in 37 DEG C, 5%CO₂Cultivated with the incubator of 95% relative humidity；

D. second day observation cell survival rate, and culture medium is changed, observe cell growth status daily later；

E. when cell growth needs to carry out passage operation to cell to converging when rate reaches 80~90%, to expand cell numberAmount, maintain the good growth conditions of cell；

F. remove supernatant, add 2mL 0.25% pancreatin, after digesting 1~2min, Microscopic observation cell rounding, cellBetween gap when becoming big, add the isometric fresh cultures of 2mL and terminate digestion, move into digestion system after softly dispelling cellIn 15mL centrifuge tubes, cell centrifugation, 150 × g, 5min, remove supernatant；

G. cell precipitation is resuspended with 5mL complete mediums, by cell 1：10 assign in 10cm culture dishes, and each ware adds 500 μL cell suspensions, each culture dish supply 10mL culture mediums, and gently cross mixes plate；

H., culture dish is steadily put back to 37 DEG C, 5%CO₂Cultivated with the incubator of 95% relative humidity.

(2) rAAV packaging

A. culture AAV-293 cells converge rate to it and reach about 80~90%；Opti-MEM is preheated in 37 DEG C of water-baths,The transfection reagents of Lipofectamine 2000 are balanced to room temperature, are shaken up before use；

B. cell first mixes 1250 μ L Opti MEM and 50 μ L Lipofectamine 2000, adds pAAV-RC9 matterThe pSNAV-BsAb plasmid 5ng prepared in 4 μ g of grain, the μ g of pHelper plasmids 8 and embodiment 3, are well mixed again, you can obtainTransfection composite needed for per ware；

C. transfection composite is added slowly in the 10cm culture dishes of culture AAV-293 cells, gently mixed；After transfection6h, transfection liquid is replaced with to fresh complete medium；

D. 72h after transfecting, the cell of the particle containing rAAV is scraped with cell and gently scraped, is collected in 15mL centrifuge tubes, 150× g centrifugations 3min collects cell, removes culture supernatant, is washed once with PBS, cell finally is resuspended with 300 μ L PBS again；

E. 37 DEG C of thermostat water baths and liquid nitrogen are prepared, by the centrifuge tube equipped with cell in liquid nitrogen and 37 DEG C of water-bath multigelationsThree times.4 DEG C, 2000 × g, 5min, cell fragment is removed, collects the cracking supernatant of the particle containing rAAV.

(3) rAAV purifying and concentration

A. 0.1 μ L Benonase enzymes, 37 DEG C of water-baths 1h, 600 × g, 4 DEG C, centrifugation are added in cracking supernatant per 1mL10min, collect supernatant；

B. PBS is added in the supernatant of collection, volume is complemented into 4mL, then with being added after 0.45 μm of membrane filtrationThe Buffer P of 1/4 viral volume, fully mix, stood overnight in 4 DEG C, 1500 × g, 4 DEG C of centrifugation 30min, abandon supernatant, collectRAAV viral pellets；

C. take 4mL Buffer S dissolvings that rAAV viral pellets are resuspended, 1400 × g, 4 DEG C of centrifugation 5min, supernatant is shiftedIn the centrifuge tube clean to one, above-mentioned steps are repeated once；

D. gained supernatant is added in super filter tube, 1400 × g centrifugation 20min, until supernatant volume narrows down to 300 μ LLeft and right, transfer it in a sterile EP pipe, then take 100 μ L Buffer S to clean concentration tube, collect rAAV diseasesPoison；

E. resin column is placed in 15mL centrifuge tubes, 150 × g centrifugation 2min, removes the sealing of bottom of the pillar, be reentered intoInto 15mL centrifuge tubes, unscrew cap, allow Buffer to add 4mL Buffer S with gravity stream, treating that liquid flows to end, allow itWith under gravity stream；

F. the rAAV virus liquids that will be obtained in step d, are drop by drop slowly added into pillar, are incorporated into resinOn；

G. take 4mL Buffer ES to add in resin column, elute rAAV virions；

H. the 4mL rAAV viral sample liquid that will be obtained, is added in super filter tube, 1400 × g centrifugation 30min, obtains about200μL rAAV.It is viral (gene therapeutic agents) to collect the rAAV-BsAb after purification finally given, is stored in -80 DEG C.

Test case one

The titre detection of rAAV-BsAb viruses

The rAAV-BsAb viruses of purifying in embodiment 4 are diluted 4000 times with PBS, then addition DNase I, 37 DEG CIt is incubated 1h；It is subsequently added into Proteinase K and reaches 300mg/L to its concentration eventually, 50 DEG C is incubated 1h.Last 95 DEG C of incubations20min, Proteinase K are inactivated, obtain rAAV genomic samples.Finally, total extension rate of rAAV-BsAb viral samplesFor 40000 times.

Standard items plasmid is subjected to gradient dilution, the copy gradient for setting standard items is 10⁶、10⁷、10⁸、10⁹、10¹⁰.Match somebody with somebodyQuantitative fluorescent PCR (QPCR) reaction system processed：

Component	Volume/
		2×SYBR Green mix	4.8μL
Sense primer (10 μm of ol/L)	0.4μL
		Anti-sense primer (10 μm of ol/L)	0.4μL
RAAV genomic samples or standard items	1.0μL
		ddH2O	3.4μL

The sequence of wherein sense primer is：Gtgcactgtg tttgctgacg, the sequence of anti-sense primer are：gaaaggagct gacaggtggt.QPCR reaction conditions are set：95 DEG C of 3min, 1 circulation；94 DEG C of 30s, 62 DEG C of 30s, 72 DEG C30s, 30 circulations；65~95 DEG C, 1 circulation (melting curve).

The drawing result of standard curve is as shown in Figure 7.It can be seen that QPCR amplification efficiencies E=10^-1/slope- 1=10^1/3.343- 1=99%, between 90%-110%；And standard curve R²=0.999, correlation is good, illustrates that QPCR primers expandEfficiency and specificity meet the requirements, and can be used for follow-up rAAV virus titers measure.The rAAV- of purifying in embodiment 4For BsAb viral samples after QPCR is detected, Ct average values are 12.45, according to standard curve and extension rate, can calculate rAAV-BsAb virus titers are 1.3 × 10¹²v.g./mL.RAAV-BsAb viruses are successfully prepared.

Test case two

BsAb detections are expressed after rAAV-BsAb virus infected cells

(1) Skeletal Muscle Cells of Mouse C2C12 is cultivated

1. the recovery of C2C12 cells

A. the C2C12 cell cryopreservation tubes to be recovered are taken out from liquid nitrogen, are immediately placed in 37 DEG C of water-baths, and shake frequentlyIt is set to thaw rapidly；

B. 9mL DMEM complete mediums are taken in 15mL centrifuge tubes, the C2C12 cells after defrosting are moved into centrifuge tube,Gently mix, 600rpm centrifugations 5min；

C. centrifugation terminates, and abandons most supernatant, adds DMEM complete medium suspension cells of the 10mL containing 10%FBS, is transferred toIn 10cm culture dishes, CO is positioned over₂Saturated humidity, 37 DEG C, 5%CO in incubator₂Concentration culture；

2. the passage of C2C12 cells

A. moved into after observing the growing state of C2C12 cells in workbench, rewarming DMEM complete mediums at 37 DEG C；

B. remove culture supernatant, add 2mL 0.25% pancreatin, after digesting 1~2min, Microscopic observation cell rounding,When intercellular gap becomes big, add the isometric fresh cultures of 2mL and terminate digestion, move digestion system after softly dispelling cellEnter in 15mL centrifuge tubes, cell centrifugation, 150 × g, 5min, remove supernatant；

C. cell precipitation is resuspended with 5mL DMEM complete mediums, cell is adjusted into concentration to 2 × 10⁵Individual/mL is seeded toIn 24 porocyte culture plates, each hole adds 1mL cell suspensions；

D., culture plate is steadily put back to 37 DEG C, 5%CO₂Cultivated with the incubator of 95% relative humidity.

(2) rAAV-BsAb infection cells

A is when C2C12 cells length to individual layer respectively with the purifying in MOI=10000 inoculum concentration infection embodiment 4RAAV-BsAb viruses, infect 4 hole cells；

B. changed after infecting 1h with new nutrient solution, 37 DEG C, 5%CO₂With culture 5~7 in the incubator of 95% relative humidityMy god；

C. virus infected cell nutrient solution is collected, 150 × g centrifugations 5min collects supernatant, added in 10kD super filter tubes, warpLow temperature is concentrated by ultrafiltration 10 times, takes 40 μ L samples after concentration to add 10 μ L 5 × albumen sample-loading buffers, 100 DEG C of high-temperature metal baths5min sample preparations are ready for use on Western-blotting analyses, and remaining is frozen in -80 DEG C.

(3) Western-blotting identifies BsAb expressions

1. SDS-PAGE electrophoresis

A. 12% separation gel, 4% concentration glue are prepared；

B. assemble after PAGE electrophoresis tanks with 10 μ L/ holes loadings, turn 100v 60min after 80v 20min；

2. transferring film

A. pvdf membrane is opened according to the size of glue, clip one, after soaking 15s in methyl alcohol, 2min is rinsed in pure water；

B. carefully short glass plate sled is fallen, concentration glue is gently scraped off, carefully peel separation calymma in transferring film buffer solution,The clip of transferring film, two blocks of foam-rubber cushions, a glass rod, filter paper and dipped are put into the enamel tray added with transfer liquid simultaneouslyFilm；

C. clip is opened makes black one side keep level.After a foam-rubber cushion is padded above a filter is covered in matPaper, filter paper is fixed on the other hand and rolls bubble therein with glass rod on the other hand；

D. separation gel is covered on filter paper, it is alignd with filter paper with hand adjustment is whole, gently bubble is rolled with glass rod, by membrane coverIn on glue, and bubble removing is removed, lid one opens filter paper on film, removes bubble, clip is closed after finally covering another foam-rubber cushion；

E. clip is put into transfer groove, makes the black flour of folder to the black flour of groove, the fine flour of folder is to the red face of groove, during electrotransferMeeting heat production, an ice chest cooling, 80v transfers 100min are put on one side of groove；

3. immune response

A. after film is soaked from bottom to top with PBS, move in the sample sack containing 15mL confining liquids, shake closing at room temperatureRinsed twice with PBST after 1h；

B. primary antibody is suitably diluted with PBST by concentration, cuts off a new sample sack, antibody-solutions are uniformly addedTo sample sack bottom surface, after transfer film is sucked into Liquid Residue with filter paper, memebrane protein is put on antibody liquid level down, lifts film fourResidual bubble is removed in angle to catch up with, and by edge sealing after sample sack wrap film, after being incubated 1h at room temperature, shakes washing three at room temperature with PBSTIt is secondary, each 10min；

C. the HRP secondary antibodies marked are suitably diluted with PBST according to the concentration of manufacturer's recommended, cuts off a new sampleProduct bag, antibody-solutions are uniformly added to sample sack bottom surface, after transfer film is sucked into Liquid Residue with filter paper, memebrane protein is faced into decentralizationResidual bubble is removed to catch up with antibody liquid level, lifting film corner, edge sealing after sample sack wrap film after being incubated 1h at room temperature, is usedPBST shakes washing three times at room temperature, each 10min；

D. develop：Chemiluminescence chromogenic reaction is carried out according to ECL kit specifications：An EP pipe is taken, is separately added into ECLDevelop the color A liquid and each 200 μ L of B liquid, is added to after well mixed on transfer film, nitrite ion is uniformly coated on into transfer film with spreading rodOn, room temperature lucifuge is incubated 10min, exposure imaging, the molecular weight of object tape and net optical density is analyzed with gel images processing systemValue.BsAb expressions are as shown in Figure 8.It can be seen that by after rAAV-BsAb virus infection, C2C12 cell successful expressions go outBsAb (swimming lane 2), illustrates the ability that rAAV-BsAb viruses can assign cell expression BsAb, and rAAV-BsAb viruses can be used as geneTherapeutic agent imports in cell and successful expression.

Test case three

Flow cytometry BsAb respectively and effector cell and target cell combination

(1) cell culture

1. the recovery of Jurkat, Raji cell

A. Jurkat, Raji cell cryopreservation tube to be recovered is taken out from liquid nitrogen, is immediately placed in 37 DEG C of water-baths, notWhen shake it is thawed rapidly；

B. 5mL RPMI-1640 complete mediums are taken in 15mL centrifuge tubes, Jurkat, Raji cell after defrosting are movedEnter in centrifuge tube, gently mix, 600rpm centrifugations 5min；

C. centrifugation terminates, and abandons most supernatant, adds 5mL RPMI-1640 complete medium suspension cells, is transferred to T25 blake bottlesIn, it is positioned over CO₂Saturated humidity, 37 DEG C, 5%CO in incubator₂Concentration culture；

2. the passage of Jurkat, Raji cell

A. moved into after observing the growing state of Jurkat, Raji cell in workbench, rewarming RPMI-1640 is complete at 37 DEG CCulture medium；

B. it is 3~5 × 10 to add new RPMI-1640 complete mediums adjustment cell density⁵Individual/mL, it is distributed into newIn Tissue Culture Flask, after micro- Microscopic observation cell state, CO is positioned over₂Saturated humidity, 37 DEG C, 5%CO in incubator₂ConcentrationContinue to cultivate.

(2) Flow cytometry BsAb respectively and effector cell and target cell combination

A. Jurkat, Raji cell each 2mL, 2000rpm centrifugation 5min of culture is taken to collect cell, FACS buffer solution washingCell is once；

B. cell, adjustment cell density to 5 × 10 is resuspended with the FACS buffer solution of appropriate volume⁶Individual/mL, then according to 90μ L/ pipes are dispensed into 1.5mL EP pipes；

C. respectively by 10 μ L infect and be uninfected by the C2C12 cells of rAAV-BsAb viruses concentration supernatant and Jurkat orRaji cells are incubated 30min under the conditions of 4 DEG C；

D. 900 μ L FACS buffer solutions are added, 2000rpm centrifugation 4min, supernatant is abandoned and is washed carefully with 1mL FACS buffer solutions againBorn of the same parents are once；

E. cell precipitation is resuspended in the antibody diluted with 100 μ L, and 30min is incubated under the conditions of 4 DEG C；

F. add after 900 μ L FACS buffer solutions after 2000rpm centrifugations 4min, abandon supernatant and washed again with 1mL FACS buffer solutionsWash cell once；

G.200 μ L FACS buffer solutions use flow cytomery after cell is resuspended.

Flow cytometry result is as shown in figure 9, wherein Jurkat cell expresses CD3 antigens, with anti-on BsAbThe display of CD3Fv fragments combines；Raji cells express CD20 antigens, are combined with anti-CD20Fv fragments display on BsAb.Tie aboveFruit shows that BsAb shows good binding ability with CD20 antigens and CD3 antigens.

Test case four

The BsAb lethal effects of Flow cytometry rAAV-BsAb mediation expression

Effector cell Jurkat and target cell Raji is prepared according to test case three, Cell viability requirement is higher than 95%；SimultaneouslyBy the method for test case two BsAb is expressed with C2C12 cells.

Raji cells are marked with Calcein AM, according to effect target than 10:1 addition Jurkat cell is incubated after 4h with extremely altogetherCell dye EthD-1 carries out cell dyeing, machine testing in last streaming.

Incubation system is divided into 5 groups：Group A is that Raji cells add culture medium to be incubated；Group B is that Raji cells add Jurkat cell to be total toIt is incubated；Group C is that Raji cells add Jurkat cell to add final concentration of 1ng/mL BsAb to be incubated altogether；Group D is that Raji cells addJurkat cell adds final concentration of 1ng/mL BsAb and is incubated altogether；Group E be Raji cells add Jurkat cell add eventually it is denseSpend and be incubated altogether for 100ng/mL BsAb.

As a result it is as shown in Figure 10：BsAb group C, group D, group E are added compared with the group B for being not added with BsAb, dead cell in system(Q1 quadrants) ratio is obviously improved, and dead cell ratio and the dosage of BsAb additions have dependence.Illustrate that BsAb canIt is effective to kill B cell lymphoma cell Raji.

Embodiment described above only expresses one or more of embodiments of the present invention, and it describes more specific and detailedCarefully, but the limitation to the scope of the claims of the present invention therefore can not be interpreted as.It should be pointed out that the common skill for this areaFor art personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to this hairBright protection domain.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

<110>The accurate medical science and technology Co., Ltd in Shenzhen

<120>Saltant type bispecific antibody and its application

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<170> SIPOSequenceListing 1.0

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Asp Ile Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser

1 5 10 15

Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser

20 25 30

Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp

35 40 45

Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro

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Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 2

<211> 123

<212> PRT

<213>Artificial sequence (Artificial Sequence)

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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser

115 120

<210> 3

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<213>Artificial sequence (Artificial Sequence)

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Asp Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

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Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Thr Thr Asp Lys Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

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Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

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Thr Thr Val Thr Val Ser Ser

115

<210> 4

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<213>Artificial sequence (Artificial Sequence)

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Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr Met

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Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Ser Leu Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 5

<211> 528

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<213>Artificial sequence (Artificial Sequence)

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

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Val His Ser Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ile Gln Ile Val

20 25 30

Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val

35 40 45

Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Phe

50 55 60

Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser

65 70 75 80

Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala

100 105 110

Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly

115 120 125

Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala

145 150 155 160

Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro

180 185 190

Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp

195 200 205

Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

210 215 220

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

225 230 235 240

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp

245 250 255

Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala

260 265 270

Ala Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Gln Ser Gly Ala

275 280 285

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

290 295 300

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg Gln Ala Pro

305 310 315 320

Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

325 330 335

Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Thr Thr Asp

340 345 350

Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu

355 360 365

Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys

370 375 380

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Glu

385 390 395 400

Gly Thr Ser Thr Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ala Asp

405 410 415

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

420 425 430

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Tyr Met

435 440 445

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

450 455 460

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

465 470 475 480

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Ser Leu Glu Ala Glu

485 490 495

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

500 505 510

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys His His His His His His

515 520 525

<210> 6

<211> 1587

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactccgac 60

tacaaagatg atgacgataa ggatatccag atcgtgctga gccagagccc cgccatcctg 120

agcgccagcc ccggcgagaa ggtgaccatg acctgccgcg ccagcagcag cgtgagctac 180

atccactggt tccagcagaa gcccggcagc agccccaagc cctggatcta cgccaccagc 240

aacctggcca gcggcgtgcc cgtgcgcttc agcggcagcg gcagcggcac cagctacagc 300

ctgaccatca gccgcgtgga ggccgaggac gccgccacct actactgcca gcagtggacc 360

agcaaccccc ccaccttcgg cggcggcacc aagctggaga tcaagcgcgg tggtggtggt 420

tctggcggcg gcggctccgg tggtggtggt tctcaggtgc agctgcagca gcccggcgcc 480

gagctggtga agcccggcgc cagcgtgaag atgagctgca aggccagcgg ctacaccttc 540

accagctaca acatgcactg ggtgaagcag acccccggcc gcggcctgga gtggatcggc 600

gccatctacc ccggcaacgg cgacaccagc tacaaccaga agttcaaggg caaggccacc 660

ctgaccgccg acaagagcag cagcaccgcc tacatgcagc tgagcagcct gaccagcgag 720

gacagcgccg tgtactactg cgcccgcagc acctactacg gcggcgactg gtacttcaac 780

gtgtggggcg ccggcaccac cgtgaccgtg agcgccgcct ccggaggtgg tggctccgac 840

gtccaactgg tgcagtcagg ggctgaagtg aaaaaacctg gggcctcagt gaaggtgtcc 900

tgcaaggctt ctggctacac ctttactagg tacacgatgc actgggtaag gcaggcacct 960

ggacagggtc tggaatggat tggatacatt aatcctagcc gtggttatac taattacgca 1020

gacagcgtca agggccgctt cacaatcact acagacaaat ccaccagcac agcctacatg 1080

gaactgagca gcctgcgttc tgaggacact gcaacctatt actgtgcaag atattatgat 1140

gatcattact gccttgacta ctggggccaa ggcaccacgg tcaccgtctc ctcaggcgaa 1200

ggtactagta ctggttctgg tggaagtgga ggttcaggtg gagcagacga cattgtactg 1260

acccagtctc cagcaactct gtctctgtct ccaggggagc gtgccaccct gagctgcaga 1320

gccagtcaaa gtgtaagtta catgaactgg taccagcaga agccgggcaa ggcacccaaa 1380

agatggattt atgacacatc caaagtggct tctggagtcc ctgctcgctt cagtggcagt 1440

gggtctggga ccgactactc tctcacaatc aacagcttgg aggctgaaga tgctgccact 1500

tattactgcc aacagtggag tagtaacccg ctcacgttcg gtggcgggac caaggtggag 1560

atcaaacatc atcaccatca tcattag 1587

Claims (10)

1. A kind of 1. saltant type bispecific antibody, it is characterised in that including be sequentially connected anti-CD20VL mutant, firstConnect peptide, anti-CD20VH mutant, the second connection peptide, anti-CD3VH mutant, the 3rd connection peptide and anti-CD3VLMutant；

   Wherein, the anti-CD20VL mutant includes the polypeptide being made up of the amino acid sequence shown in SEQ ID No.1, instituteState the polypeptide that anti-CD20VH mutant includes being made up of the amino acid sequence shown in SEQ ID No.2, the anti-CD3VHMutant includes the polypeptide that is made up of the amino acid sequence shown in SEQ ID No.3, the anti-CD3VL mutant include byThe polypeptide of amino acid sequence composition shown in SEQ ID No.4, the saltant type bispecific antibody is single-chain antibody.
2. 2. saltant type bispecific antibody according to claim 1, it is characterised in that the saltant type bispecific antibodyAlso include Flag label proteins and His label proteins, the Flag label proteins mark is in the anti-CD20VL mutantN-terminal, C-terminal of the His labels egg mark in the anti-CD3VL mutant.
3. 3. saltant type bispecific antibody according to claim 1, it is characterised in that the saltant type bispecific antibodyIncluding:(a) amino acid shown in the polypeptide being made up of the amino acid sequence shown in SEQ ID No.5, (b) and SEQ ID No.5The polypeptide of sequence composition with least 98% homology and with the active polypeptide of the saltant type bispecific antibody, or (c),The polypeptide being made up of the amino acid sequence shown in SEQ ID No.5, wherein one or more amino acid are lacked, substituted or increasedObtain and with the polypeptide of saltant type bispecific antibody activity.
4. 4. saltant type bispecific antibody according to claim 1, it is characterised in that the saltant type bispecific antibodyIncluding：(a), the polypeptide that the polynucleotide encoding being made up of the nucleotide sequence shown in SEQ ID No.6 obtains, (b) and SEQThe polynucleotide encoding that the polynucleotides of nucleotide sequence composition shown in ID No.6 have at least 98% homology is obtained and hadHave a polypeptide of saltant type bispecific antibody activity, or (c), be made up of the nucleotide sequence shown in SEQ ID No.6Polynucleotides, wherein one or more bases are lacked, substituted or increased obtained polynucleotide encoding and obtained and with describedThe polypeptide of saltant type bispecific antibody activity.
5. 5. a kind of saltant type bispecific antibody expressing gene, it is characterised in that for encoding mutant type bispecific antibody, instituteAnti-CD20VL mutant, the first connection peptide, the anti-CD20VH that stating saltant type bispecific antibody includes being sequentially connected dash forwardVariant, the second connection peptide, anti-CD3VH mutant, the 3rd connection peptide and anti-CD3VL mutant；

   Wherein, the anti-CD20VL mutant includes the polypeptide being made up of the amino acid sequence shown in SEQ ID No.1, instituteState the polypeptide that anti-CD20VH mutant includes being made up of the amino acid sequence shown in SEQ ID No.2, the anti-CD3VHMutant includes the polypeptide that is made up of the amino acid sequence shown in SEQ ID No.3, the anti-CD3VL mutant include byThe polypeptide of amino acid sequence composition shown in SEQ ID No.4, the saltant type bispecific antibody is single-chain antibody.
6. 6. saltant type bispecific antibody expressing gene according to claim 5, it is characterised in that the saltant type is double specialHeterogenetic antibody expressing gene includes：(a) shown in, the nucleotide sequence shown in SEQ ID No.6, (b) and SEQ ID No.6Nucleotide sequence has the nucleotide sequence of at least 98% homology, or the nucleotide sequence shown in (c), SEQ ID No.6, itsMiddle one or more bases are lacked, substituted or increased obtained nucleotide sequence.
7. 7. a kind of recombinant expression carrier, it is characterised in that have the saltant type as described in any one of claim 5~6 double specialProperty antibody expressing genes.
8. 8. a kind of host cell, it is characterised in that there is the saltant type bispecific as described in any one of claim 5~6 to resistBody expressing gene.
9. A kind of 9. gene therapeutic agents, it is characterised in that including adeno-associated virus and carry in the adeno-associated virus asSaltant type bispecific antibody expressing gene described in any one of claim 5~6.
10. 10. the saltant type bispecific antibody or as described in any one of claim 5~6 as described in any one of Claims 1 to 4Application of the saltant type bispecific antibody expressing gene in anti-tumor drug is prepared.