CN110330566A

CN110330566A - A kind of immunoglobulin that the bispecific with dual variable domains combines

Info

Publication number: CN110330566A
Application number: CN201910505149.8A
Authority: CN
Inventors: 姜东成
Original assignee: Nanjing Huayan Biotechnology Co Ltd
Current assignee: Nanjing Huayan Biotechnology Co Ltd
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2019-10-15

Abstract

The immunoglobulin for the bispecific combination with dual variable domains that the invention discloses a kind of, the recombination immunoglobulin includes polypeptide chain V from aminoterminal to c-terminus_b(X) n-VLa-CL, wherein V_bTo match the single-domain structure that the polypeptide chain of combination being made of multiple amino acid formed with target, X is connexon, n >=0, VLa is the variable domains of light chain, CL is light chain constant domain, and a is the first antigen that bispecific combines, and b is the second antigen that bispecific combines.The immunoglobulin that bispecific with dual variable domains of the invention combines, steric hindrance is small, has stronger tumor tissues penetration power；Molecular weight and natural antibody are close, long half time；Molecular structure bilateral symmetry, is not present mispairing；Since single domain antibody is merged with the light chain of natural antibody, the affinity of heavy chain will not be impacted, therefore good with the affinity of two antigen.

Description

A kind of immunoglobulin that the bispecific with dual variable domains combines

Technical field

The invention belongs to immunoglobulin technical fields, are related to the system of the recombination immunoglobulin of bispecific antigen binding It is standby and application thereof, and in particular to a kind of immunoglobulin that the bispecific with dual variable domains combines.

Background technique

Bispecific antibody (Bispecific antibody) is containing there are two the immunoglobulins of different antigen binding sites Molecule.Due to its specificity and bi-functional, increases the binding affinity to target, become the research in antibody engineering field Hot spot has broad application prospects in the fields such as immunotherapy of tumors and autoimmunity disease.Due to not depositing under natural conditions In bispecific antibody, can only be prepared by specific process such as genetic engineering transformations.Due to antibody light chain weight corresponding to its The pairing of chain is the interchain disulfide bond formed based on the cysteine in two polypeptide chains, and pairing process is simultaneously non-specific, different Random combine between peptide chain results in the pairing from two kinds of not light chains and heavy chain of synantigen, theoretically there is 10 kinds of combinations Form, wherein most by-products for being paired into the pairing of light chain heavy chain mistake, so the core of bispecific antibody technology at present The heart is all around the mismatch problems for solving two kinds of not light chains of synantigen and heavy chain.

In the past few years, researcher develops the bispecific antibody of various structures.Current bispecific antibody Two classes can be divided into, one kind has been free from antibody Fc domain, and representative has BiTE technology, DART technology and TandAbs technology. BiTE technology be the antibody variable region for combining CD3 positive T cell light chain with heavy chain in conjunction with tumor cell surface antigen The light chain of antibody is connected in series with heavy chain, and being formed can be in combination with the more of the bispecific of CD3 positive T cell and tumour cell combination Peptide.DART technology is to combine the heterodimer antibody formed by two polypeptide chains, and structure is by an antibody variable region VH and VL sequence connect to be formed with VL the and VH sequence of another antibody variable region respectively.In addition, in the C-terminal of two polypeptide chains Cysteine is introduced, interchain disulfide bond is formed by cysteine, improves the stability of product.TandAbs technology is by two Molecule peptide chain reversely matches the homodimer molecule of formation, structure Fva-Fvb-Fvb-Fva.Such bispecific combines The shortcomings that antibody is that molecular weight is small, half-life short, for example, by using " BLINCYTO " half-life period of marketed drug of BiTE technology Have 2-3 hours, administration mode single administration is needed with special infusion device successive administration 24-48 hours.It has another disadvantage that Due to do not contain antibody Fc structure, so be unable to the antibody-dependant of mediate antibody cell mediated cytotoxicity (ADCC, (antibody-dependent cell-mediated cytotoxicity, ADCC), refer to expression IgGFc receptor NK cell, Macrophage and neutrophil leucocyte etc., by with the IgG that has been incorporated into the target cell surfaces such as virus infected cell and tumour cell The Fc section of antibody combines, and kills the effect of these target cells), the cytotoxic effect (CDC (complement of Complement Dependent Dependent cytotoxicity, CDC), through specific antibody in conjunction with cell membrane surface corresponding antigens, form compound And activating complement classical pathway, be formed by membrane attack complex to target cell play cracking effect) etc. antibody mediated immunities killing activity Function.From molecular structure, antibody that this antibody structure cannot at last truly.

The technology used in bispecific antibody platform containing the area Fc has: (1) solving heavy chain mispairing by the mutation area Fc Problem solves light chain mismatch problems by being mutated or exchanging constant region of light chain and the first constant region of heavy chain.Representative technology has " Knobs-into-holes " and " Cross Mab " technology of Roche Holding Ag's exploitation, Zymeworks company develop Azymetric technology, Wuhan You Zhiyou company exploitation " YBODY " technology, Trion/Fresenius company " Triomab " Technology.Such technology reduces mispairing to a certain extent, but shortcoming is also evident from.Those skilled in the art all know, antibody The area Fc mediates ADCC, and the immune functions such as CDC, ADCP, these immune functions are the important sides that antibody molecule realizes its pharmacy function Formula.Human immunity responsing reaction is extremely complex system, and carrying out mutation to Fc structural domain will cause unknown immune side reaction, Bring huge uncertainty.Above-mentioned technical proposal does not all disclose the correlative study and safety data of immune response.Secondly, light The combination active zone that the exchanging of the mutation of chain or constant region of light chain (CL) and the first constant region of heavy chain (CH1) also gives antigen comes Uncertainty.For example, (the The Journal of Immunology 1998,160:2802- such as SherisL.Morrison 2808) it has studied heavy chain of antibody and exchanges influence to antibody function with light chain variable region, it is indicated that heavy chain of antibody and light chain variable region Exchange can reduce the combination power of antibody and antigen.(2) another kind of technical solution has through the C-terminal or N-terminal in heavy chain of antibody Connect the second antigen binding domain.Representative technical solution has: " DVD-Ig " technology of Abbvie company exploitation, Merrimack " IgG-scFv " and bank of company step " FIT-Ig " technology of biology exploitation.The common feature of such technology is molecular structure or so Symmetrically, mispairing is solved.One critical defect be heavy chain connect the second antigen variable region, caused by steric hindrance largely effect on The combination activity of heavy chain variable region and antigen, one side those skilled in the art note, antibody in conjunction with antigentic specificity can Become area, play the variable region that primary specificity combination is heavy chain, the research of SANDRA.J etc. is also indicated that in antibody and antigen Specific binding event in that play a decisive role is antibody heavy chain variable region (area VH) (J Immunol, vol.139:4135- 4144.No.12.December15.1987).Even there was only heavy chain in camel and the intracorporal antibody of shark class animal, light chain is natural Missing, is also single domain antibody.One is also reported in patent (number of patent application 201510008045.8, US7183076) application Kind there is the bispecific antibody of common light chain, such bispecific antibody has common light chain, with two not synantigens What specific binding was leaned on is the variable region of heavy chain, it is seen then that is played a major role in antibody with antigen binding is the variable region of heavy chain. On the other hand, above-mentioned technical proposal is to merge the Fab segment of the second antigen in the variable region (area VH) of heavy chain, will certainly be to heavy chain The combination of variable region and the first antigen causes very big influence, interferes the realization of antibody function.And it is not also given in original application Out with the data of two specific antigen affinity.Furthermore the antibody molecule amount of such technical solution is anti-much larger than natural Body, steric hindrance caused by macromolecule is difficult to penetrate solid tumor mass, and macromolecule easily causes immunogenicity, brings Safety issue.

Inventor thinks that an ideal bi-specific antibody molecule is (1) containing the area natural antibody Fc, can mediate The antibody such as ADCC, CDC can lower immunogene in vivo containing natural antibody Fc simultaneously to the immunologic cytotoxicity activity of target Property；(2) molecular weight and natural antibody are close, therefore long half time, and immunogenicity is low, good to solid tumor penetrability；(3) molecule pair Claim, mispairing is not present, is produced convenient for purifying and technique amplification；(4) there is high affinity with two antigens.Second antigen binding Area will not cause space steric effect to the first antigen, interfere the combination activity of the first antigen binding domain.

Summary of the invention

Goal of the invention: being directed to the deficiencies in the prior art, and the object of the present invention is to provide one kind to have dual can be changed The immunoglobulin that the bispecific of structural domain combines, single domain is merged with the light chain of natural antibody, effectively solution mesh The problem of existing influence heavy chain affinity of preceding bispecific antibody and mispairing.It is a further object of the present invention to provide in one kind State the application for the immunoglobulin that the bispecific with dual variable domains combines.

Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:

A kind of immunoglobulin that the bispecific with dual variable domains combines, includes two polypeptide chains, wherein One polypeptide chain is V from aminoterminal to c-terminus_b(X) n-VLa-CL, wherein Vb is the single domain of combination of matching with target Structure, X are connexon, and n >=0, VLa are the variable domains of light chain, and CL is light chain constant domain, and a is bispecific combination The first antigen, b be bispecific combine the second antigen.

The Article 2 polypeptide chain for the immunoglobulin that the bispecific combines is VHa- from aminoterminal to c-terminus CH1-Fc, wherein VHa is the variable domains of heavy chain, and CH1 is the first constant domain of heavy chain constant region.

The polypeptide chain institute shape that the described single-domain structure of combination that matches with target is made of for one multiple amino acid At structural domain.

The structural domain is the variable region of single domain antibody, antigen, cell factor, cytokine receptor, cell surface egg It is white, chemotactic factor (CF), chemokine receptors.

The single-domain structure is the extracellular domain of antigen.

The CH1 structural domain and CL structural domain is wild type or saltant type structural domain.

The Fc structural domain is wild type or saltant type structural domain.

The single-domain structure refers to the combination that can match with target, is rolled over by the polypeptide chain that multiple amino acid form The folded polypeptide domain formed.

The single domain antibody, source are the serum of camellid or shark.

The polypeptide chain that the connexon X is made of multiple identical or different amino acid.

A kind of immunoglobulin that the bispecific with dual variable domains combines, includes two polypeptide chains, wherein One polypeptide chain is VHa-CH1-Fc- (X) from aminoterminal to c-terminus_n-V_b, wherein V_bFor the list for the combination that matches with target Domain structure；X is connection unit, n >=0；VHa is the variable domains of heavy chain, and CH1 is the first constant structure of heavy chain constant region Domain.

The Article 2 polypeptide chain for the immunoglobulin that the bispecific combines is VLa- from aminoterminal to c-terminus CL, wherein VLa is the variable domains of light chain, and CL is light chain constant domain.

The described single-domain structure for matching combination with target is single domain antibody.

The single domain antibody, a source are the serum of camellid or shark.

The immunoglobulin that the bispecific with dual variable domains combines is in preparation for treating the mankind Application in disease medicament.

The utility model has the advantages that compared with prior art, what the bispecific with dual variable domains of the invention combined exempts from Epidemic disease globulin, steric hindrance is small, has stronger tumor tissues penetration power；Molecular weight and natural antibody are close, long half time；Point Minor structure bilateral symmetry, is not present mispairing, can carry out large-scale industrialized production；Since single domain antibody is light with natural antibody Chain fusion, will not impact the affinity of heavy chain, therefore good with the affinity of two antigen.

Detailed description of the invention

Fig. 1 is the first structural schematic diagram for the immunoglobulin that there is the bispecific of dual variable domains to combine；

Fig. 2 is second of structural schematic diagram of the immunoglobulin that there is the bispecific of dual variable domains to combine.

Specific embodiment

The present invention is described further combined with specific embodiments below.Unless otherwise indicated, the art used in the present invention Language is the general meaning in this field, is known to those skilled in the art.

" single-domain structure " used in the present invention, which refers to, to be rolled over as a polypeptide as composed by multiple amino acid by space The folded unit with biological function formed.The characteristics of single-domain structure of the invention is to tie with target (antigen) specificity It closes.Such as single-domain structure can be the variable region of single domain antibody, then the extracellular domain that such as single-domain structure can be antigen.

The extracellular domain of antigen refers to that antigen is exposed to the part of cell surface, can be special with the antigenic determinant of antibody The opposite sex combines to trigger the structural domain of a series of signal access and signal transduction.

Single domain antibody of the present invention, also known as nano antibody or heavy chain antibody are the blood from camellid and shark A kind of antibody isolated in clear.Single domain antibody is only made of heavy chain, and antigen binding domain is only one by hinge area and Fc Area connection single domain, and this antigen binding domain separate from antibody after still have combine antigen function.

" VHH structural domain " is also known as the varistructure of the combination antigen of single domain heavy chain antibody (nano antibody, heavy chain antibody) Domain.It is that heavy-chain variable domains (VH) and light chain in conventional 4 chain antibodies of difference can using term " VHH structural domain " purpose Structure changes domain (VL).VHH structural domain being capable of the independent combination antigen of specificity.

" Fab structural domain " refers to comprising antibody's light chain variable region (VL), constant region of light chain (CL) and heavy chain variable region (VH), again The first constant region of chain (CH1)

Term " Fc structural domain " refers to by two heavy chains second, the albumen that third constant domain is composed of by disulfide bond Matter segment.

Embodiment 1

This patent thinks that a good bispecific antibody should have following characteristics:

1) molecular weight and natural antibody are close, have the half-life period similar with natural antibody.

2) with natural antibody Fc structure (Fc structure is without mutation), ADCC, the immunologic cytotoxicities activity such as CDC can be mediated.

3) molecular structure is symmetrical, and mispairing is not present, can technique amplification and large-scale production；

4) there is good solid tumor penetration capacity.

Current bispecific antibody technology is all unable to satisfy above-mentioned requirements.

For example, be representative without antibody Fc district using BiTE technology bispecific antibody technology (such as BiTE technology, DART technology and TandAbs technology) advantage be that antibody molecule amount is small, penetration power is strong.The disadvantage is that antibody molecule half-life short, gives Clinical application brings great inconvenience.It is needed using the administration mode single administration of the BLINCYTO of marketed drug of BiTE technology With special infusion device successive administration 24-48 hours, clinical application is greatly limited.

In the bispecific antibody technology containing antibody Fc district, the technical tactic of use has: (1) in heavy chain of antibody N-terminal The second antigen binding domain is merged, representative technical solution has " DVD-Ig " technology of Abbott, and bank steps the " FIT- of biology Ig " technology, the advantage of this kind of technology are that solve mismatch problems.The disadvantage is that immunogenicity caused by molecular weight excessive (250KD) The safety issue of increase and the penetration capacity of solid tumor, another is maximum the disadvantage is that in the side of heavy chain of antibody N-terminal fusion Formula can the steric hindrance caused by antibody and the first antigen binding, influence to have been acknowledged with the binding force of the first antigen, inventor Fab segment (the Fab of the second antigen is combined in the fusion of heavy chain of antibody N-terminal₂) binding force of antibody and the first antigen can be subtracted significantly It is weak.(2) another technical approach is to merge the second antigen binding domain, such as " IgG- of Merck & Co., Inc. in heavy chain of antibody C-terminal Technical solution in scFv " technology and Chinese patent application 201480016032.9 (PCT/US2014/028731), this kind of skill The shortcomings that art, is: the penetration capacity of safety issue and solid tumor that immunogenicity caused by molecular weight is excessive increases；In antibody Heavy chain C-terminal (area Fc) fusion causes unpredictable influence to the immunologic cytotoxicity active (ADCC, CDA function) that Fc is mediated.(3) another A kind of technical approach is mutation to be carried out to antibody light chain constant region (area CL) heavy chain constant region (area CH1) or to antibody chain constant Area (area CL) heavy chain constant region (area CH1) exchange solves weight to heavy chain of antibody Fc region mutation to solve light chain mismatch problems Chain mismatch problems.Representative technical solution has " the CrossMab+Knobs into holes " of Roche Holding Ag, Zymeworks " Azymetric " technology of company and " YBODY " technology of You Zhiyou biotech firm.The shortcomings that this kind of technical solution, is reduction of Mismatch rate but it cannot be fully solved mispairing, heavy chain mutant can cause antibody immunologic cytotoxicity activity (such as ADCC, CDC activity) The influence and risk that cannot be predicted exchange antibody light chain constant region (area CL) and the first constant region of heavy chain (area CH1) or prominent Become the combination activity that can reduce antibody and antigen, influences the biological function of antibody.Such as SherisL.Morrison etc. is ground Study carefully and points out that heavy chain of antibody exchanges combination power (the The Journal of that can reduce antibody and antigen with light chain variable region Immunology 1998,160:2802-2808).

The present embodiment uses and blends the single-domain structure specifically bound with target (antigen) with natural antibody light chain Technical solution, invented the bispecific antibody that single-domain structure-natural antibody blends.Bispecific antibody tool of the invention Have that molecular weight is close with natural antibody, long half-lift, low immunogenicity；Molecular machinery is symmetrical, and mispairing is not present；With two antigens All there is good affinity；The feature of the immunologic cytotoxicity activity of mediate antibody is capable of in the area Fc with natural antibody.

Specifically, the immunoglobulin that the bispecific with dual variable domains combines, there are two types of basic frameworks for tool Structure, as shown in Figure 1, the first is comprising two polypeptide chains, and first: from aminoterminal to c-terminus Vb- (X) n-VLa-CL, In, Vb is the single-domain structure of combination of matching with target, and X is connection unit, and n >=0, VLa are the variable domains of light chain, CL For light chain constant domain, a is the first antigen that bispecific combines, and b is the second antigen that bispecific combines.Article 2: From aminoterminal to c-terminus VHa-CH1-Fc, wherein VHa is the variable domains of heavy chain, and CH1 is the first perseverance of heavy chain constant region Constant domain.

Second as shown in Fig. 2, also include two polypeptide chains, first: from aminoterminal to c-terminus include polypeptide chain VHa-CH1-Fc- (X) n-Vb, wherein Vb is the single-domain structure of combination of matching with target；X is connection unit, n >=0；VHa For the variable domains of heavy chain, CH1 is the first constant domain of heavy chain constant region.Article 2 polypeptide chain: from aminoterminal to carboxylic Cardinal extremity includes VLa-CL, wherein VLa is the variable domains of light chain, and CL is light chain constant domain.

Single-domain structure refer to target (antigen) specific binding, function is formed by by a polypeptide chain folding Structural domain；It is preferred that the variable region (antigen binding domain) of single domain antibody.The feature of variable region one of single domain antibody is from single domain antibody Still there is the function in conjunction with antigen after separation, it can be in Camelidae (such as alpaca, camel etc.) and shark class animal blood serum. Preparation method are as follows:

(1) antigen is immune.Be immunized using intravenous injection antigen, every 1~2 week it is immune once, exempt from for process three to four times Epidemic disease starts to acquire camel/alpaca jugular vein whole blood, detects serum titer, continue to be immunized if potency is low.

(2) separating peripheral blood mononuclear cells (PBMC) from camel/alpaca peripheral blood, after extract RNA, with the side RT-PCR Method specific amplification alpaca antibody heavy chain variable region (VHH) segment；And use two step connection methods by antibody heavy chain variable region piece Section is connect with phagemid vector obtains recon, and electricity obtains VHH Antibody geometric mean titer after turning competent E.coli TG1；And it uses The dilution method of counting measures antibody library storage capacity, random picked clones sequence verification antibody library diversity.

(3) screening of antibody library.Phage antibody library is obtained using helper phage.The immune pipe of antigen coat, is added VHH Phage antibody goes to infect TG1 again after incubation by the bacteriophage that washing-elution obtains.Continue packaging second to take turns needed for elutriation Bacteriophage repeats first round panning process.After the titre for eluting bacteriophage has the raising of about 2 orders of magnitude, stop washing in a pan Choosing.Do Phage Elisa identification monoclonal sequence.

(4) single domain antibody humanization.Using a variety of Humanized strategies, transplants such as CDR and/or analyzed back based on homologous structure The antibody library building being mutated again carries out humanization to antibody with screening.

Bispecific antibody of the invention is merged by single-domain structure with monoclonal antibody light chain, wherein monoclonal antibody is Known to those skilled in the art, refer to origin source nature especially derive from animal B-cells secretion by two light chains and two The antibody that the molecular weight of heavy chain composition is about 150KD, preferably IgG.

The monoclonal antibody that the present invention and single-domain structure blend, the structure with natural monoclonal antibody can be Wild type may be saltant type, and mutation includes by monoclonal antibody function region mutation, such as light chain variable region VL, light chain Constant region CL, heavy chain variable region CH, heavy chain the first constant region CH1, heavy chain second constant region CH2, heavy chain third constant region CH3 or Hinge area.

Single-domain structure of the invention can be blended directly with monoclonal antibody, can also be merged by connexon X, even Connect the polypeptide that sub- X is made of multiple identical or different amino acid.Such as connexon of the invention can be single domain antibody Hinge area.

Bispecific antibody of the invention can be in combination with two similar and different targets.The target include but It is not limited to antigen, cell factor, cytokine receptor, cell surface protein, chemotactic factor (CF), chemokine receptors etc..Specifically Including but not limited to: immunologic test point target spot PD-1, PDL-1, XO40 (CD134), LAG-3, TIM-3, CTLA-4, KIR, GITR, VISTA, IDO-1,4-1BB (CD137), TDO2, PSGL-1, B7-H3 (CD276), CD27 etc..Interleukins IL1A, IL1B、IL2、IL3、IL4、IL5、IL6、IL7、IL8、IL9、IL10、IL11、IL12A、IL12B、IL13、IL14、IL15、 IL16、IL17A、IL17F、IL8、IL19、IL20、IL22、IL23、IL24、IL25、IL26、IL27、IL28、IL29、IL30、 IL31, IL32, IL33 etc. and interleukin-1 receptor ILR family.Interferon IFN-α (the various hypotype IFN-α including IFN-α 1, IFN-α 2, IFN-α 3 etc.) and IFN-β, IFN-γ, IFN- λ 1 (IL-29), IFN- λ 2 (IL-28a) and IFN- λ (IL-28b)； Tumor necrosis factor TNF-alpha, TNF-beta, TNFSF, CD95L, TRAIL, VEGI；TNF receptor TNF-R1, TNF-R2；Colony-stimulating Factor granulocyte-macrophage colony stimutaing factor (GM-CSF), Granulocyte macrophage-colony stimulating factor (M-CSF), grain are thin Born of the same parents' colony stimulating factor (G-CSF), erythropoietin(EPO) (EPO), stem cell factor (SCF) and thrombopoietin (TPO) etc.；Chemotactic cytokine includes the CXC chemotactic cytokine of α subfamily, if IL-8 is the representative of α subfamily system； The CC chemotactic cytokine of β subfamily, if monocyte chemoattractant protein-1 (MCP-1) is the representative of β subfamily；γ subfamily Chemotactic cytokine (C chemotactic cytokine), if Lymphotactin (lymphotactin) is γ subfamily It represents；Transforming growth factor includes TGFα, TGF β (TGF β 1, TGF β 2, TGF β 3) etc.；Growth factors and receptors include that epidermis is thin It is the intracellular growth factor (EGF), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), intravascular Skin cell growth factor receptor (VEGFR), fibroblast growth factor (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF16、FGF7、FGF8、FGF9、FGF10、FGF11、FGF12、FGF13、FGF14、FGF15、FGF16、FGF17、FGF19、 FGF20, FGF21, FGF22, FGF23) and its receptor family (FGFR), nerve growth factor (NGF)；Nerve growth factor by Body NGFR, TNF-R I (CD120a), TNF-R II (CD120b), CD40, CD95, CD27, CD30 and human medullary cell surface Active antigen Fas (CD95)；The growth factor (PDGF) in blood platelet source and its receptor (PDGFR) etc..

Embodiment 2

A method of the immunoglobulin that the bispecific with dual variable domains combines is prepared, specifically: training It supports host cell of the invention and recycles the bispecific antibody from culture.Antibody can be conventional by isolating and purifying etc. Method purifies to obtain from culture medium, and conventional method is known to a person skilled in the art, such as in-depth filtration, affinity chromatography, Anion chromatography, cation chromatography, affinity chromatography remove virus filtration, ultrafiltration etc..

The host cell that can be used for inventing is preferably mammalian cell, more preferably Chinese hamster ovary celI, HK293 cell, BHK Cell, HeLa cell, COS cell, most preferably Chinese hamster ovary celI.The present invention can also be used plant cell, insect cell, Escherichia coli, Saccharomycete etc..

The building of this method expression vector and the conversion of host cell can be used conventional cell biology and molecule raw Object and technique for gene engineering carry out.Conventional cell biology refers to people from this field with molecular biology and technique for gene engineering Biology techniques known to member, such as DNA sequence dna synthesis, electrotransformation, vector construction, determination of activity etc..

The immunoglobulin that bispecific with dual variable domains of the invention combines can be used for specific antibody All application fields.Especially it is used to prepare the antibody drug for the treatment of human diseases.Including treating various entity tumors and blood The endocrine system diseases such as liquid tumour, autoimmune disease, diabetes, various inflammation and anaphylactia, nervous system disease The various human diseases such as disease, graft versus host disease.Including to the of the invention double special of object application therapeutically effective amount Property antibody molecule binding molecule, inhibit object in growth of tumour cell.It can be with using bi-specific antibody molecule of the invention The preferred cancer of prevention and/or treatment includes generally having the cancer of response to immunization therapy.Medicable preferred cancer it is non- Restrictive example includes lung cancer, oophoroma, colon and rectum carcinoma, melanoma (such as malignant mela noma of transfer), kidney Cancer, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngocarcinoma, uterine neck Cancer, corpus uteri tumor and osteosarcoma, osteocarcinoma, cancer of pancreas, cutaneum carcinoma, prostate cancer, skin or intraocular malignant melanoma, uterus Cancer, cancer of the anal region, carcinoma of testis, carcinoma of fallopian tube, carcinoma of endometrium, carcinoma of vagina, Hodgkin's disease, non_hodgkin lymphoma, cancer of the esophagus, It is carcinoma of small intestine, internal system cancer, thyroid cancer, parathyroid carcinoma, adrenal, soft tissue sarcoma, carcinoma of urethra, chronic or acute Leukaemia, including it is acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic Lymphocytic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, maincenter Nervous system (CNS) tumour, primary CNS lymphoma, tumor vessel generation, tumor of spine, brain stem glioma, pituitary gland Tumor, Kaposi sarcoma, epidermis shape cancer, squamous cell carcinoma, t cell lymphoma, ambient induced cancer, the cancer including Induced by Asbestos The combination of disease and the cancer.

Above-mentioned human diseases includes that can be prevented and/or be treated using bi-specific antibody molecule of the invention to the object The unrestricted example of preferred autoimmune disease include that chronic lymphocytic thyroiditis, thyroid function are high Into, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anaemia be with atrophic gastritis, empsyxis Nephritic syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute special hair The organ specific autoimmune diseases such as property multiple neuritis；And systemic loupus erythematosus, rheumatoid arthritis, rigid spine Inflammation, psoriasis, systemic vasculitis, chorionitis, day born of the same parents' sore, dermatomyositis, autoimmune hemolytic anemia, thyroid gland itself are exempted from Epidemic disease, mixed connective tissue disease, ulcerative colitis, adolescent diabetes, essential thrombocythemia purpura and many kinds of skins The systemic autoimmune diseases such as skin disease, chronic liver disease, polyneuritis.

Above-mentioned human diseases includes that can be prevented and/or be treated using bi-specific antibody molecule of the invention to the object Preferred infectious virus and bacterium, inflammation, endocrine system, the non-limit of cardiovascular and cerebrovascular and other and immune-related disease The example of property processed includes hepatitis virus (first, second, third), influenza virus, herpesviral, giardia lamblia, malaria, Leishmania, gold Staphylococcus aureus, Pseudomonas aeruginosa, herpesviral, adenovirus, influenza virus, arboviruse, echovirus, rhinovirus, Ke Sa Odd virus, coronavirus, Respiratory Syncytial Virus(RSV), mumps virus, rotavirus, measles virus, rubella virus, carefully Small virus, vaccinia virus, dengue fever virus, papillomavirus, contagiosum, poliovirus, rabies viruses, insect-borne diseases Malicious encephalitis viruses, Chlamydia, rickettsia bacterium, mycobacteria, staphylococcus, streptococcus, pneumococcus, meningococcus and Gonococcus, klebsiella, mycetozoan, thunder Salmonella, pseudomonad, Legionnella, corynebacterium diphtheriae, salmonella, gemma bar Bacterium, cholera bacteria, tetanolysin, clostridium botulinum, bacillus anthracis, yersinia pestis, Leptospira and Lyme disease bacterium.Arthritis, Osteoarthritis, juvenile chronic arthritis, septic arthritis, Lai Mushi arthritis, adjuvant arthritis, SpA, Crohn's disease, inflammatory bowel disease, hyperlipidemia, hypertension, diabetes, primary biliary hardening, hemolytic anemia, pernicious disease Disease, heart failure, myocardial infarction, polyadenous volume defect, adult respiratory distress syndrome, arthropathia psoriatica, ulcerative colitis Inflammatory arthropathy, intestines source property synovitis, Yersinia ruckeri and salmonella correlation arthropathy, osteoporosis, SpA, Atheromatosis/artery sclerosis, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, line Property IgA disease, autoimmune hemolytic anemia, pernicious anemia,juvenile, chronic mucocutaneous candidiasis, giant cell artery Inflammation, primary sclerotic hepatitis, hidden source property lupoid hepatitis, acquired immunodeficiency disease syndrome, acquired immunity The diseases such as defect related disease, hepatitis B, hepatitis C, common variability immune deficiency, graft-versus-host.

Embodiment 3

A kind of preparation medicine of the immunoglobulin combined containing the bispecific with dual variable domains of the invention Compositions, the pharmaceutical composition medium or carrier can be water for injection, normal saline solution or 5% glucose solution.Medicine Compositions may include for changing, keep or save such as the pH of composition, osmotic pressure, viscosity, transparency, color, Infiltration property, smell, aseptic, stability, dissolution or rate of release, adsorptivity or infiltrative formulation materials.Suitable preparation material Material includes but is not limited to amino acid: such as glycine, glutamine, asparagine, arginine or lysine)；Antioxidant: Such as ascorbic acid, sodium sulfite or sodium hydrogensulfite；Buffer system: such as acetate, bicarbonate, Tris-HCl, lemon Hydrochlorate, phosphate, histidine, glycine or other organic acids；Filler: such as mannitol or glycine；Chelating agent: such as second Ethylenediamine tetraacetic acid (EDTA)；Polyalcohol: sorbierite, mannitol, sucrose；Albumen: such as seralbumin, gelatin or immunoglobulin)； Emulsifier；Surfactant: such as Pluronic, PEG, sorbitol ester, polysorbate (such as polysorbate20, poly- mountain Pears alcohol ester 80), tromethamine, lecithin, cholesterol；Stabilizing reinforcer (sucrose or sorbierite).

The medicine composition includes but is not limited to freeze drying powder injection, liquid drugs injection, preliminary filling injection.Wherein, liquid drugs injection is 1-500mL, preferably 1-250mL, more preferable 1-100mL, most preferably 5-50mL.

In one embodiment, the immunoglobulin that the bispecific with dual variable domains of the invention combines It is designed to target two antigens on the same target cell to adjust and believe with human diseases generation and treatment-related cell Number conduction, which can be tumour cell, be also possible to immunocyte such as T cell or B cell or other and mankind's disease Disease occurs and treatment-related target cell.

In another embodiment, the immune globulin that the bispecific with dual variable domains of the invention combines It is white to be designed to target cell with extracellularly free antigen to adjust and human diseases occurs and treatment-related cell signal Conduction, target cell can be that tumour cell is also possible to immunocyte such as T cell or B cell or other and human diseases occur And treatment-related target cell.

In another embodiment, the immunoglobulin of the bispecific combination with dual variable domains of the invention Be designed to two extracellularly free antigen adjust and human diseases occurs and treatment-related cellular signal transduction.

Again in another embodiment, the immune globulin of the bispecific combination with dual variable domains of the invention It is white to be designed to two different target cell antigens of targeting to adjust and human diseases generation and treatment-related cell signal Conduction, target cell can be that tumour cell is also possible to immunocyte or it occurs with human diseases and treatment-related target is thin Born of the same parents.

Embodiment 4

1, the screening of single domain antibody

1.1 camels or alpaca are immune

Immune ANTIGEN DESIGNThe requires molecule sufficiently large.Average every 5-10kD just has an epitope, therefore molecule is too small More peptide or proteins are difficult have an epitope.It is exogenous strong.If the antigen and intracorporal substance of machine is just the same or phase It is seemingly difficult to cause the immune response of body.Structure is complicated as far as possible.Simple duplicate substance is without immunogenicity.

Intravenous injection antigen is immunized, and every 1~2 week immune primary, and process three to four times immune, starts to acquire alpaca neck Venous whole detects serum titer, continues to be immunized if potency is low.

The building of 1.2 antibody libraries

Separating peripheral blood mononuclear cells (PBMC) from alpaca peripheral blood, after extract RNA, with RT-PCR method specificity Expand alpaca antibody heavy chain variable region (VHH) segment；And use two step connection methods by antibody heavy chain variable region segment and phagocytosis Grain carrier connection obtains recon, and electricity obtains VHH Antibody geometric mean titer after turning competent E.coli TGI；And it is counted using dilution Method measures antibody library storage capacity, random picked clones sequence verification antibody library diversity.

The screening of 1.3 antibody libraries

Phage antibody library is obtained using helper phage.The immune pipe of antigen coat, is added VHH phage antibody, is incubated for It goes to infect TG1 again by the bacteriophage that washing-elution obtains afterwards.Continue bacteriophage needed for packaging second takes turns elutriation, repeats first Take turns panning process.After the titre for eluting bacteriophage has the raising of about 2 orders of magnitude, stop elutriation.It is Phage Elisa Identify monoclonal sequence.

1.4 single domain antibody humanizations

Using a variety of Humanized strategies, the antibody library structure of back mutation is transplanted and/or analyzed based on homologous structure such as CDR It builds and humanization is carried out to antibody with screening.

2. building, expression and the characterization of anti-PDL-1/VEGFR2 bispecific antibody

The building of 2.1 antibody molecules

It is merged using PDL-1 single domain antibody variable region (area VHH) with anti-vegf R2 monoclonal antibody light chain, building obtains anti-PDL-1/ VEGFR2 bispecific antibody.Anti-vegf R2 monoclonal antibody uses Lei Molu monoclonal antibody (ramuricumab)；PDL-1 single domain antibody uses 1 In method screen and obtain out of camel body.PDL-1 single domain antibody variable region (area VHH) is melted with anti-vegf R2 antibody light chain It closes, obtains polypeptide chain VHH_PDL-1-VL_VEGFR2-CL(SEQ ID NO.1)；Anti- PDL-1/VEGFR2 bispecific antibody heavy chain uses Lei Molu monoclonal antibody VH_VEGFR2-CH1-Fc(SEQ ID NO.2)

The expression and characterization of 2.2 antibody molecules

(1) HEK-293 cell is placed in 5% CO₂In constant-temperature table, isothermal vibration culture, determine its cell density and Survival rate.It is in exponential phase using growth, cell of the survival rate greater than 97% transfects；Cell is directly blended into without centrifugation Cell density is diluted to 3 × 10 by KOP293 culture solution⁶A/milliliter；Shaking flask is placed in 5% CO₂In constant-temperature table, 37 DEG C, 120rpm isothermal vibration culture.

(2) it transiently transfects

100mL cell suspension is transfected, the sterile centrifugation tube of two 15mL is prepared.5mL is added in a wherein centrifuge tube KPM and the 50 sterile Plasmid DNA of μ g, gently piping and druming mixes；Another centrifuge tube is taken, 5mL KPM and 500 μ l TA-293 is added and turns Transfection reagent, gently piping and druming mixes；Liquid in two centrifuge tubes is mixed, gently piping and druming mixes, and stands 10 minutes at room temperature and makes Standby plasmid-carrier complexes out；Cell is taken out from constant-temperature table, and plasmid-carrier complexes are uniformly added into cell suspension And with hand even, CO is put back to₂It is cultivated in constant-temperature table.

(3) product expression and detection

500 μ l, 293 expression of cellular proteins reinforcing agent (KE-293) can be added after 24 hours in transfection, to increase product expression Amount；The expression quantity of antibody is measured after continuing culture 6 days；Then, supernatant is purified by Protein A affinity chromatography, Content of monomer and stability are measured by technologies such as mass spectrometry, SDS-PAGE.Bispecific tetravalent antibody can be steady as the result is shown Fixed expression.The activity of the combination to PD-1 and VEGF is studied by ELISA and Biacore measuring method, shows the double special of generation Property tetravalent antibody can be in combination with PD-1 and VEGF, and the signal path both blocked simultaneously plays anti-tumor activity.

Embodiment 5

Identify building, expression and the characterization of TNF-α and the tetravalence bispecific antibody of IL-17, detailed process is as follows:

The building of 1 antibody molecule

Merged using TNF-α single domain antibody variable region (area VHH) with anti-IL-17 monoclonal antibody light chain, building obtain anti-tnf-alpha/ IL-17 tetravalence bispecific antibody.Anti-tnf-alpha single domain antibody screens to obtain using the method in 1.Anti- IL-17 monoclonal antibody amino acid Sequence derives from patent WO2007/070750.TNF-α single domain antibody variable region (area VHH) is melted with anti-IL-17 antibody light chain It closes, obtains polypeptide chain VHH_TNF-α-VL_IL-17-CL(SEQ ID NO.3)；Anti-tnf-alpha/IL-17 bispecific antibody heavy chain uses Amino acid sequence VH in patent WO2007/070750_IL-17-CH1-Fc(SEQ ID NO.4)。

The expression and characterization of 2 antibody molecules

Antibody molecule expression uses HEK-293 cell, and the operating method in use 2.2 carries out transient expression, uses Protein A affinity chromatography purifies target product, will be purified using the technical methods such as mass spectrometry, SDS-PAGE, HPLC Obtained target protein is characterized.The results show that bispecific tetravalent antibody can stablize expression.By ELISA and Biacore measuring method is active to study the combination to PDL-1 and VEGFR, shows that the bispecific tetravalent antibody of generation can be same When combine PDL-1 and VEGFR, and block their activity simultaneously.

Embodiment 6

Identify building, expression and the characterization of the tetravalence bispecific antibody of VEGF and PD-1, detailed process is as follows:

The building of 1 antibody molecule

VEGFR1 (vegf receptor 1) extracellular domain belongs to antigenic structure, can specifically bind with VEGF.VEGFR1 2nd area Ig (VEGFR1D2) of (vegf receptor 1) extracellular domain is merged with anti-PD-1 antibody light chain, building obtain anti-vegf/ PD-1 tetravalence bispecific antibody.The amino acid sequence of VEGFR1D2 is shown in SEQ ID NO.5, and anti-PD-1 monoclonal antibody is using receiving Wu Dankang (nivolumab).VEGFR1D2 is merged with anti-PD-1 antibody light chain, obtains polypeptide chain VEGFR1D2-VL_PD-1-CL(SEQ ID NO.6)；Anti-vegf R/PD-1 bispecific antibody heavy chain is using receiving military monoclonal antibody heavy chain VH_PD-1-CH1-Fc(SEQ ID NO.7)。

The expression and characterization of 2 antibody molecules

Antibody molecule expression uses HEK-293 cell, and the operating method in use 2.2 carries out transient expression, uses Protein A affinity chromatography purifies target product, will be purified using the technical methods such as mass spectrometry, SDS-PAGE, HPLC Obtained target protein is characterized.The results show that bispecific tetravalent antibody can stablize expression.Determination of activity is studied pair The combination activity of PD-1 and VEGF shows that the bispecific tetravalent antibody of generation can be in combination with PD-1 and VEGF, and simultaneously Block their activity.

Embodiment 7

Identify building, expression and the characterization of the tetravalence bispecific antibody of her2 and her3, detailed process is as follows:

The building of 1 antibody molecule

It is merged using her3 single domain antibody variable region (area VHH) with anti-her2 monoclonal antibody light chain, building obtains anti-her2/her3 Tetravalence bispecific antibody.Anti- her3 single domain antibody screens to obtain using the method in 1.Anti- her2 monoclonal antibody amino acid sequence uses Herceptin amino acid sequence.Her3 single domain antibody variable region (area VHH) is merged with anti-her2 antibody light chain, obtains polypeptide Chain VHH_her3-VL_her2-CL(SEQ ID NO.8)；Anti- her2/her3 bispecific antibody heavy chain uses the amino of Herceptin Acid sequence VH_her2-CH1-Fc(SEQ ID NO.9)。

The expression and characterization of 2 antibody molecules

Antibody molecule expression uses HEK-293 cell, and the operating method in use 2.2 carries out transient expression, uses Protein A affinity chromatography purifies target product, will be purified using the technical methods such as mass spectrometry, SDS-PAGE, HPLC Obtained target protein is characterized.The results show that bispecific tetravalent antibody can stablize expression.By ELISA and Biacore measuring method is active to study the combination to her2 and her3, shows that the bispecific tetravalent antibody of generation can be simultaneously In conjunction with her2 and her3, and their activity is blocked simultaneously.

Sequence table

<110>Nanjing Hua Yan Bioisystech Co., Ltd

<120>a kind of immunoglobulin that the bispecific with dual variable domains combines

<130> 100

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 352

<212> PRT

<213>VHHPDL-1-VLVEGFR2-CL amino acid sequence (Artificial)

<400> 1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Arg

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Lys Asn Thr Val Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Ser Phe

85 90 95

Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser Gly Ala Phe Gln Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

130 135 140

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

145 150 155 160

Cys Arg Ala Ser Gln Gly Ile Asp Asn Trp Leu Gly Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu

180 185 190

Asp Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr

195 200 205

Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Phe Ala Val Tyr

210 215 220

Phe Cys Gln Gln Ala Lys Ala Phe Pro Pro Thr Phe Gly Gly Gly Thr

225 230 235 240

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

245 250 255

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

260 265 270

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

275 280 285

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

290 295 300

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

305 310 315 320

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

325 330 335

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345 350

<210> 2

<211> 446

<212> PRT

<213>VHVEGFR2-CH1-Fc amino acid sequence (Artificial)

<400> 2

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Leu Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 3

<211> 340

<212> PRT

<213>VHHTNF- α-VLIL-17-CL amino acid sequence (Artificial)

<400> 3

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr Tyr Ser Ser Asn

20 25 30

Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Ala Ile Tyr Thr Gly Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Gly Leu Gly Leu Val Glu Arg Thr Phe Arg Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser Asp Ile Val Met Thr Gln Thr

115 120 125

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

130 135 140

Arg Ser Ser Arg Ser Leu Val His Ser Arg Gly Asn Thr Tyr Leu His

145 150 155 160

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys

165 170 175

Val Ser Asn Arg Phe Ile Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

180 185 190

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

195 200 205

Val Gly Val Tyr Tyr Cys Ser Gln Ser Thr His Leu Pro Phe Thr Phe

210 215 220

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

225 230 235 240

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

245 250 255

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

260 265 270

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

275 280 285

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

290 295 300

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

305 310 315 320

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

325 330 335

Arg Gly Glu Cys

340

<210> 4

<211> 449

<212> PRT

<213>VHIL-17-CH1-Fc amino acid sequence (Artificial)

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Asn Pro Met Tyr Gly Thr Thr Asp Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Asp Tyr Phe Thr Gly Thr Gly Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Leu

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 5

<211> 104

<212> PRT

<213>VEGFR1D2 amino acid sequence (Artificial)

<400> 5

Leu Glu Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu

1 5 10 15

Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro

20 25 30

Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro

35 40 45

Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg

50 55 60

Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu

65 70 75 80

Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu

85 90 95

Thr His Arg Gln Thr Asn Thr Ile

100

<210> 6

<211> 318

<212> PRT

<213>VEGFR1D2-VLPD-1-CL amino acid sequence (Artificial)

<400> 6

Leu Glu Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu

1 5 10 15

Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro

20 25 30

Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro

35 40 45

Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg

50 55 60

Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu

65 70 75 80

Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu

85 90 95

Thr His Arg Gln Thr Asn Thr Ile Glu Ile Val Leu Thr Gln Ser Pro

100 105 110

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

115 120 125

Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro

130 135 140

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr

145 150 155 160

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

165 170 175

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

180 185 190

Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val

195 200 205

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

210 215 220

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

225 230 235 240

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

245 250 255

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

260 265 270

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

275 280 285

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

290 295 300

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

305 310 315

<210> 7

<211> 440

<212> PRT

<213>VHPD-1-CH1-Fc amino acid sequence (Artificial)

<400> 7

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

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

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

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

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

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

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 8

<211> 340

<212> PRT

<213>VHHher3-VLher-2-CL amino acid sequence (Artificial)

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Pro Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Tyr Ser Arg His

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Thr Asn Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Lys Pro Gly Trp Met Ala Thr Leu Ala Trp Ser Asp Trp Phe Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Gly Asp Ile

115 120 125

Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

130 135 140

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala

145 150 155 160

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

165 170 175

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

180 185 190

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

195 200 205

Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe

210 215 220

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

245 250 255

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

260 265 270

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

275 280 285

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

290 295 300

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

305 310 315 320

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

325 330 335

Arg Gly Glu Cys

340

<210> 9

<211> 451

<212> PRT

<213>VHher-2-CH1-Fc amino acid sequence (Artificial)

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

Claims

1. a kind of immunoglobulin that the bispecific with dual variable domains combines, which is characterized in that the recombination Immunoglobulin includes two polypeptide chains, wherein a polypeptide chain is V from aminoterminal to c-terminus_b(X) n-VLa-CL, wherein V_bFor the single-domain structure for the combination that matches with target, X is connexon, and n >=0, VLa are the variable domains of light chain, and CL is light Chain constant domain, a are the first antigen that bispecific combines, and b is the second antigen that bispecific combines.

2. the immunoglobulin that the bispecific according to claim 1 with dual variable domains combines, feature It is, the Article 2 polypeptide chain for the immunoglobulin that the bispecific combines, is VHa-CH1- from aminoterminal to c-terminus Fc, wherein VHa is the variable domains of heavy chain, and CH1 is the first constant domain of heavy chain constant region.

3. the immunoglobulin that the bispecific according to claim 1 or 2 with dual variable domains combines, special Sign is, the single-domain structure that combination is matched with target is formed by one by the polypeptide chain that multiple amino acid form Structural domain.

4. the immunoglobulin that the bispecific according to claim 3 with dual variable domains combines, feature It is, variable region of the structural domain for single domain antibody, antigen, cell factor, cytokine receptor, cell surface protein, Chemotactic factor (CF), chemokine receptors.

5. the immunoglobulin that the bispecific according to claim 1 or 3 with dual variable domains combines, special Sign is that the single-domain structure is the extracellular domain of antigen.

6. the immunoglobulin that the bispecific according to claim 1 or 2 with dual variable domains combines, special Sign is that the CH1 structural domain and CL structural domain are wild type or saltant type structural domain.

7. the immunoglobulin that the bispecific according to claim 2 with dual variable domains combines, feature It is, the Fc structural domain is wild type or saltant type structural domain.

8. the immunoglobulin that the bispecific according to claim 1 or 2 with dual variable domains combines, special Sign is that the single-domain structure refers to the combination that can match with target, the polypeptide chain folding being made of multiple amino acid The polypeptide domain of formation.

9. the immunoglobulin that the bispecific according to claim 4 with dual variable domains combines, feature It is, the single domain antibody, source is the serum of camellid or shark.

10. the immunoglobulin that the bispecific according to claim 1 with dual variable domains combines, feature It is, the polypeptide chain that the connexon X is made of multiple identical or different amino acid.

11. a kind of immunoglobulin that the bispecific with dual variable domains combines, which is characterized in that the recombination Immunoglobulin includes two polypeptide chains, wherein a polypeptide chain is VHa-CH1-Fc- (X) from aminoterminal to c-terminus_n-V_b, In, V_bFor the single-domain structure for the combination that matches with target；X is connection unit, n >=0；VHa is the variable domains of heavy chain, CH1 is the first constant domain of heavy chain constant region.

12. the immunoglobulin that the bispecific according to claim 11 with dual variable domains combines, special Sign is that the Article 2 polypeptide chain for the immunoglobulin that the bispecific combines is VLa-CL from aminoterminal to c-terminus, Wherein, VLa is the variable domains of light chain, and CL is light chain constant domain.

13. the immunoglobulin that the bispecific according to claim 11 or 12 with dual variable domains combines, It is characterized in that, the single-domain structure, refers to the combination that can match with target, the polypeptide chain being made of multiple amino acid Fold the polypeptide domain formed.

14. the immunoglobulin that the bispecific according to claim 11 or 12 with dual variable domains combines, It is characterized in that, the single-domain structure of the combination that matches with target is single domain antibody.

15. the immunoglobulin that the bispecific according to claim 14 with dual variable domains combines, special Sign is that the single domain antibody a, source is the serum of camellid or shark.

16. the immunoglobulin that the bispecific according to claim 12 with dual variable domains combines, special Sign is, the polypeptide chain that the connexon X is made of multiple identical or different amino acid.

17. the immune ball that the described in any item bispecifics with dual variable domains of claim 1,2,11,12 combine Albumen is in preparation for treating the application in human diseases drug.