CN106986939A - anti-PD-1 and TEM-8 bispecific antibody and application thereof - Google Patents

Abstract

The present invention relates to an anti-PD-1 and TEM-8 bispecific antibody, or a variant thereof, or a functional fragment thereof, comprising: a domain that specifically recognizes and binds to an immune cell surface antigen PD-1, comprising the heavy chain variable region of an anti-PD-1 specific antibody (anti-PD-1 VH); and a domain that specifically recognizes and binds to tumor endothelial marker TEM-8, comprising the heavy chain variable region of an anti-TEM-8 specific antibody (anti-TEM-8 VH). The bispecific antibodies against PD-1 and TEM-8 have significantly improved anti-tumor targeting, reduced risk of therapeutic side effects, and at the same time have significantly improved effectiveness. The experimental results show that compared with antibodies of other tumor endothelial markers, the bispecific antibody formed by combining the anti-PD-1 antibody and the anti-TEM-8 antibody has obvious synergistic effect, namely the curative effect of the bispecific antibody is statistically and significantly improved compared with the curative effect of two single antibodies.

Description

Anti- PD-1 and TEM-8 bispecific antibodies and its application

Technical field

The invention belongs to biomedicine field, be related to used in research and treatment use to PD-1 and TEM-8 simultaneously With the bispecific antibody of high-affinity or its variant or functional fragment, and further to anti-involved by coding The nucleic acid molecules of body or its variant or functional fragment, the table for expressing the antibody or its variant or functional fragment Up to the preparation method of carrier and host cell, and the antibody or its variant or functional fragment.Present invention also offers Using the method for the bispecific antibody or its variant or functional fragment of the present invention, the therapeutic composition comprising it, with And its for strengthening the function of immunocyte, the cell-mediated immune response of up-regulation and for treating immune cell function dysfunctional Illness, such as tumour immunity, and the purposes for treating cancer.

Background technology

Bispecific antibody refers to the artificial antibody containing 2 species-specific antigen binding sites, its can in target cell and Bridge is erected between functional molecular (cell), the immune response with guidance quality is excited, is one kind of genetic engineering antibody.Double spies Heterogenetic antibody turns into the focus in antibody engineering field, is had broad application prospects in the immunization therapy of tumour.At present, The preparation of this antibody mainly has three kinds of methods：Chemical coupling method, slush pump underlayer method and genetic engineering antibody prepare method, its Middle genetic engineering antibody preparation method is the most frequently used, also most advanced.In general, to be segmented into two big for bispecific binding protein molecule Class：One class is that, containing Fc areas, another kind of to be a lack of Fc areas, the latter is typically less than immunoglobulin and IgG containing Fc areas is double Specific molecular.Fc areas are conducive to purifying bispecific antibody using the flow for IgG molecules set up, and And its dissolubility and stability can be improved.In addition, Fc areas also have an impact to the Fc- effector functions mediated, this is probably that some are controlled Treat and apply required additive effect, the cytotoxicity (ADCC) of such as antibody-dependant, complement combine (CDC) and because it has more Long half-lift caused by the cyclic process of big molecular weight and Fc neonatal receptors (FcRn) mediation.These functions can pass through The means of genetic engineering are further utilized, for example, by eliminating ADCC and/or CDC while retaining long half-lift.Compared to it Under, the bispecific binding protein molecule for lacking Fc areas is completely dependent on its antigen binding capacity to play treatment effect.

Programmed cell death factors 1 (programmed death 1, PD-1) are a kind of main in activating immune cell The Inhibitory receptor of upper expression.In tumor patient, negativity adjustment signal, ginseng are immunized by PD-1 high expression mediate T cell With negative regulator (Liu J, et al., the Plasma cells from multiple myeloma of tumor immunity patients express B7-H1(PD-L1)and increase expression after stimulation with IFN-γ and TLR ligands via a MyD88-, TRAF6-, and MEK-dependent pathway [J] .Blood, 2007,110 (1)：296-304).

Programmed cell death factors ligand 1 (programmed death 1-ligand, PD-L1) and programmed cell Death factors part 2 (programmed death 2-ligand, PD-L2) be PD-1 binding partners (Keir ME, et al., PD-1 and its ligands in tolerance and immunity [J] .Annu Rev Immunol, 2008,26： 677-704).After PD-L1 is combined with PD-1 extracellular regions, die body is suppressed by the immunity receptor tyrosine of PD-1 cytoplasmic region afterbodys, The phosphatidylinositol3 3 kinase of CD3 (CD28) inductions and the activity of protein kinase B are lowered, suppresses turning for T cell proliferation-associated genes Record and promote t cell proliferation；Protein-tyrosine-phosphatase 1 is also recruited simultaneously, makes signaling molecule dephosphorylation in connection, from And suppress Proliferative Activated and function (Jin H T, et al., Role of PD-1 in regulating the T cell of T cell Immunity [J] .Curr Top Microbiol Immunol, 2011,350 (1)：17-37).

T cell is chief component (Ahmadzadeh M, et al., the Tumor antigen- of immune system specific CD8 T cells infiltrating the tumor express high levels of PD-1 and Are functionally impaired [J] .Blood, 2009,114 (8)：1537-1544).PD-1 is in neoplasm invasiveness lymph High expression in cell, tumor specific T cells and the related T cell of some damages (Quezada SA, et al., Exploiting CTLA-4, PD-1 and PD-L1 to reactivate the host immune response Against cancer [J] .Br J Cancer, 2013,108 (8)：1560-1565), its part PD-L1 is thin in kinds of tumors There is expression in born of the same parents' (such as all kinds of non-Lymphatic System tumours, such as melanoma, liver cancer, stomach cancer, clear-cell carcinoma), show PD-1/PD- L signal path may participate in tumor immune escape (Simeone E, et al., Immunomodulating antibodies in the treatment of metastatic melanoma：The experience with anti-CTLA-4, anti- CD137 and anti-PD1 [J] .J Immunotoxicol, 2012,9 (3)：241-247).Therefore, PD-1/PD-L is blocked Anticancer therapy can be effectively carried out by suppressing immune response in tumor environment.Over the past two years, U.S. clinical tumour annual meeting Exciting clinical test is probably exactly the black whirlwind that PD-1 anticancrins bring.The clinical anti-cancer that PD-1 antibody is brought Effect is unprecedented.It can control the cancer progression of 50% cutaneum carcinoma patient, cure 10% or so skin carninomatosis People.For obstinate non-small cell lung cancer patient, medically feel simply helpless for many years, but PD-1 antibody has and faced to 24% patient Bed control effect.Due to the effect of its broad spectrum anticancer, to kidney, stomach cancer, breast cancer, carcinoma of urinary bladder, leukemia, head and neck cancer, intestinal cancer and brain The clinical efficacy of the cancers such as knurl is also among experiment of clinical the second stage of or three phases.

Tumor endothelial mark (tumor endothelia marker, TEM 1-9) is by tumor blood vessels Chrotoplast and the gene expression progress gene series winding comparison analysis (serial for closing on Normal tissue vascular endothelial cell Analysis gene express, SAGE) find 9 in the specific expressed gene of tumor endothelial cell or albumen (St Croix B, et a1., Genes expressed in human tumor endothelium [J] .Science (Wash DC),2000,289(5482):1197-1202).TEM-8 is one kind of TEM families, compared with other TEM and VEGFR, TEM- 8 express more special in tumor vascular endothelium, exist in tumor vessel, and seldom or are not present in normal structure.More attach most importance to Want, TEM-8 is then accredited as a kind of acceptor of anthrax toxin.TEM-8 has important biological function in itself, high The TEM-8 of expression can promote the adhesion and migration of cell, but the extracellular region protein that these effects can be recombinated is blocked (Hotchkiss KA,et al.,TEM-8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen.Exp Cell Res,2005,305(2):133-144)；Meanwhile, one of tumor-infiltrated and necessary cell factor of angiogenic growth-IL-1 β can be bright Aobvious induction TEM-8 expression (Rmali KA, et al., Upregulation of tumor endothelial marker-8 by interleukin-1βand its impact in IL-1βinduced angiogenesis.Int J Mol Med, 2004,14(1):75-80).In addition, for TEM-8 anti-tumour antibody sample albumen, i.e. people TEM-8 extracellular regions and human IgG FC The fusion protein TEM-8 FC of fragment, which have, significantly suppresses tumour growth activity and antitumous effect, and it is one to illustrate TEM-8 Potential anti-tumor target (Duan H F, et al., Antitumor activities of TEM8-Fc:an engineered antibody-like molecule targeting tumor endothelial marker 8.J Natl Cancer Inst, 2007,99(2):1551-1555)。

Although PD-1 antibody drugs are because it targets immunologic test point and there is powerful broad spectrum anticancer to live to kinds of tumors Property, but simultaneously there is also a variety of potential side effects, and although single target antibody therapy initially there are certain treatment Effect, would generally produce the treatment resistance caused by the up-regulation of other signal transduction cascades in subsequent treatment, it is therefore necessary to PD-1 antibody drug antitumor targeting ability ofs are further improved, the risk for the treatment of side effect is reduced, while improving validity.And with The fast development of computer science, Computer-Aided Drug Design turns into the brand-new technology of research and development new drug in recent years, Substantially accelerate the speed and efficiency of new drug design.Pharmaceutical grade protein research based on protein steric structure is extremely paid attention to, egg The continuous parsing of white matter structure, PDB data constantly increase, and make it possible CAD pharmaceutical grade protein.

The content of the invention

The defect existed for PD-1 antibody drugs in the prior art, the technical problems to be solved by the invention are to provide one Plant the new PD-1 antibody drugs with the active anticancer significantly improved.By substantial amounts of screening test and optimizing research, this hair A person of good sense have been surprisingly found that a kind of with the anti-tumor target tropism significantly improved, the treatment side effect risk of reduction, simultaneously Anti- PD-1 and TEM-8 bispecific antibody with the validity significantly improved.Most of all, test result indicates that, with The antibody of other tumor endothelial marks is compared, and anti-PD-1 antibody and anti-TEM-8 antibody are combined to be formed double special Property antibody generate obvious synergy, i.e. its curative effect have compared to the curative effect of two kinds of monospecific antibodies it is aobvious on statistical significance Write and improve, the prior art institute before this is the present invention is unforeseen.

The technical scheme that the present invention is used to solve above-mentioned technical problem is as follows：

A kind of anti-PD-1 and TEM-8 bispecific antibodies or its variant or its functional fragment, it includes：

A. specific recognition and immune cell surface antigenic PD-1 domain is combined, it includes anti-PD-1 specificity and resisted The weight chain variable district (anti-PD-1 VH) of body；And

B. specific recognition and tumor endothelial mark TEM-8 domain is combined, it includes anti-TEM-8 specificity and resisted The weight chain variable district (anti-TEM-8 VH) of body.

Preferably, the specific recognition and combination immune cell surface antigenic PD-1 domain are anti-by what is be sequentially connected Light chain variable district (anti-PD-1 VL), weight chain variable district (anti-PD-1 VH), hinge area and the Fc fragments of PD-1 specific antibodies (resist PD-1CH2-CH3) constitute, or by the light chain (anti-PD-1 VL-CL) and heavy chain (anti-PD-1 of one group of anti-PD-1 specific antibody VH-CH1- hinge areas-CH2-CH3) composition, or light chain (anti-PD-1 VL-CL) by two groups of anti-PD-1 specific antibodies and again Chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) is constituted, or by light chain variable district (the anti-PD-1 of anti-PD-1 specific antibodies VL) constituted with weight chain variable district (anti-PD-1 VH).

Preferably, the specific recognition and combination tumor endothelial mark TEM-8 domain are anti-by what is be sequentially connected Light chain variable district (anti-TEM-8 VL), weight chain variable district (anti-TEM-8 VH), hinge area and the Fc pieces of TEM-8 specific antibodies Section (anti-TEM-8 CH2-CH3) composition, or by the light chain (anti-TEM-8 VL-CL) and heavy chain of one group of anti-TEM-8 specific antibody (anti-TEM-8 VH-CH1- hinge areas-CH2-CH3) is constituted, or by light chain (the anti-TEM-8 of two groups of anti-TEM-8 specific antibodies VL-CL) constituted with heavy chain (anti-TEM-8 VH-CH1- hinge areas-CH2-CH3), or by the light chain of anti-TEM-8 specific antibodies Variable region (anti-TEM-8 VL) and weight chain variable district (anti-TEM-8 VH) composition.

According to the preferred embodiment of the present invention, the anti-PD-1 and TEM-8 bispecific antibodies or its change Body or its functional fragment include：

A ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specificity Light chain (anti-PD-1 VL-CL) and heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) composition of antibody；And

B ' specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is by one group of anti-TEM-8 specificity Light chain (anti-TEM-8 VL-CL) and heavy chain (anti-TEM-8 VH-CH1- hinge areas-CH2-CH3) composition of antibody.

Preferably, the specific recognition and the domain with reference to immune cell surface antigenic PD-1 are known with the specificity Such as domain that is other and combining tumor endothelial mark TEM-8 one or more to be located at hinge by one or more disulfide bond The disulfide bond of sequence.

According to the preferred embodiment of the present invention, the anti-PD-1 and TEM-8 bispecific antibodies or its change Body or its functional fragment include：

A " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specificity Light chain (anti-PD-1 VL-CL) and heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) composition of antibody；And

B " specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is by the anti-TEM-8 that is sequentially connected Light chain variable district (anti-TEM-8 VL), weight chain variable district (anti-TEM-8 VH), hinge area and the Fc fragments of specific antibody (resist TEM-8 CH2-CH3) composition.

Preferably, the specific recognition and the domain with reference to immune cell surface antigenic PD-1 are known with the specificity Such as domain that is other and combining tumor endothelial mark TEM-8 one or more to be located at hinge by one or more disulfide bond The disulfide bond of sequence.

According to the preferred embodiment of the present invention, the anti-PD-1 and TEM-8 bispecific antibodies or its change Body or its functional fragment include：

A " ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by the anti-PD-1 that is sequentially connected Light chain variable district (anti-PD-1 VL), weight chain variable district (anti-PD-1 VH), hinge area and Fc fragments (the anti-PD- of specific antibody 1CH2-CH3) constitute；And

B " ' specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is special by one group of anti-TEM-8 Property antibody light chain (anti-TEM-8 VL-CL) and heavy chain (anti-TEM-8 VH-CH1- hinge areas-CH2-CH3) composition.

Preferably, the specific recognition and the domain with reference to immune cell surface antigenic PD-1 are known with the specificity Such as domain that is other and combining tumor endothelial mark TEM-8 one or more to be located at hinge by one or more disulfide bond The disulfide bond of sequence.

According to the preferred embodiment of the present invention, the anti-PD-1 and TEM-8 bispecific antibodies or its change Body or its functional fragment include：

A " " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is special by two groups of anti-PD-1 Property antibody light chain (anti-PD-1 VL-CL) and heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) composition；And

B " " specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is special by two groups of anti-TEM-8 Property antibody light chain variable district (anti-TEM-8 VL) and weight chain variable district (anti-TEM-8 VH) composition.

Preferably, each anti-PD-1 in the specific recognition and combination immune cell surface antigenic PD-1 domain The CH3 of the heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) of specific antibody respectively with the specific recognition and knot Close weight chain variable district (the anti-TEM-8 of each anti-TEM-8 specific antibodies in tumor endothelial mark TEM-8 domain VH) connected by chemical bond.

Preferably, described anti-PD-1 and/or the TEM-8 bispecific antibodies or its variant or its functional fragment It is chimeric antibody, humanized antibody or human antibody.

Preferably, the specific recognition and combine PD-1 domain in weight chain variable district amino acid sequence such as SEQ ID NO:1st, shown in 5,9 or 25；The amino acid sequence of light chain variable district such as SEQ ID NO:3rd, shown in 7,11 or 27.

Preferably, the specific recognition and combine TEM-8 domain in weight chain variable district amino acid sequence such as SEQ ID NO:13rd, shown in 17,21 or 29；The amino acid sequence of light chain variable district such as SEQ ID NO:15th, 19,23 or 31 institute Show.

Preferably, the ammonia of the heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) of the anti-PD-1 specific antibodies Base acid sequence is SEQ ID No:33, nucleotides sequence is classified as SEQ ID No:34；The light chain of the anti-PD-1 specific antibodies is (anti- PD-1 VL-CL) amino acid sequence be SEQ ID No:35, nucleotides sequence is classified as SEQ ID No:36；It is described by connecting successively Light chain variable district (anti-PD-1 VL), weight chain variable district (anti-PD-1 VH), hinge area and the Fc of the anti-PD-1 specific antibodies connect The specific recognition of fragment (anti-PD-1CH2-CH3) composition and the domain for combining immune cell surface antigenic PD-1 Amino acid sequence is SEQ ID No:37, nucleotides sequence is classified as SEQ ID No:38.

Preferably, the heavy chain (anti-TEM-8 VH-CH1- hinge areas-CH2-CH3) of the anti-TEM-8 specific antibodies Amino acid sequence is SEQ ID No:39, nucleotides sequence is classified as SEQ ID No:40；The light chain of the anti-TEM-8 specific antibodies The amino acid sequence of (anti-TEM-8 VL-CL) is SEQ ID No:41, nucleotides sequence is classified as SEQ ID No:42；It is described by successively The light chain variable district (anti-TEM-8 VL) of the anti-TEM-8 specific antibodies of connection, weight chain variable district (anti-TEM-8 VH), hinge area The specific recognition and combination tumor endothelial mark TEM-8 knot constituted with Fc fragments (anti-TEM-8 CH2-CH3) The amino acid sequence in structure domain is SEQ ID No:43, nucleotides sequence is classified as SEQ ID No: 44；It is described specific by anti-TEM-8 The specific recognition and knot of light chain variable district (anti-TEM-8 VL) and weight chain variable district (anti-TEM-8 VH) composition of antibody The amino acid sequence for closing tumor endothelial mark TEM-8 domain is SEQ ID No:45, nucleotides sequence is classified as SEQ ID No:46。

On the other hand, present invention also offers one kind coding anti-PD-1 and TEM-8 bispecific antibodies or its change The nucleic acid molecules of body or its functional fragment, either a kind of expression vector comprising the nucleic acid molecules or one kind include institute State the host cell of expression vector.

Preferably, the specific recognition and combine PD-1 domain in weight chain variable district nucleotide sequence such as SEQ ID NO:2nd, shown in 6,10 or 26；The nucleotide sequence of light chain variable district such as SEQ ID NO:4th, shown in 8,12 or 28.

Preferably, the specific recognition and combine TEM-8 domain in weight chain variable district nucleotide sequence such as SEQ ID NO:14th, shown in 18,22 or 30；The nucleotide sequence of light chain variable district such as SEQ ID NO:16th, 20,24 or 32 institute Show.

Another further aspect, present invention also offers a kind of pharmaceutical composition, it is double special that it includes the anti-PD-1 and TEM-8 Property antibody or its variant or its functional fragment, the either nucleic acid molecules or the expression vector, or the host Cell.Preferably, described pharmaceutical composition also includes pharmaceutically useful carrier and/or auxiliary material；Preferably, described pharmaceutical composition is also Including other activating agents, such as chemotherapeutics, such as Tegafur, fluorouracil and oxaliplatin；Cancer adjuvant therapy medicament, such as Thymosin alpha 1, thymopeptide-5, Calciumlevofolinate, Granisetron and Tropisetron etc.；Cytotoxic agent, such as Ali's statin, calicheamicin, Maytansine and times carcinomycin etc.；Immunocyte, such as NK cells, T cell and DC-CIK cells etc..The formulation of described pharmaceutical composition Can be liquid injection formulation or solid suppository formulation.

Another aspect, present invention also offers the anti-PD-1 and TEM-8 bispecific antibodies or its variant or its work( Energy property fragment, described nucleic acid molecules, described expression vector or described host cell are preparing treating cancer, infectious disease Application in the medicine of disease or inflammatory disease.Specifically, wherein the cancer is selected from stomach cancer, carcinoma of testis, uterine cancer, fallopian tubal It is cancer, carcinoma of endometrium, cervical carcinoma, carcinoma of vagina, cancer of the esophagus, carcinoma of small intestine, thyroid cancer, parathyroid carcinoma, melanoma, kidney, preceding Row gland cancer, breast cancer, colon cancer, lung cancer, osteocarcinoma, cancer of pancreas, cutaneum carcinoma, incidence cancer, skin or intraocular chromoma, uterus Cancer, oophoroma, the carcinoma of the rectum, adrenal, cancer of the anal region, vaginal orifice cancer, carcinoma of urethra, carcinoma of penis, carcinoma of urinary bladder, kidney or carcinoma of ureter, kidney Broad-mouthed receptacle for holding liquid cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non_hodgkin lymphoma, the cancer of internal system, soft tissue Sarcoma, the neoplasm of central nervous system, primary central nervous system lymphoma, tumor vessel generation, spinal cord axle tumour, brain Dry glioma, pituitary adenoma, Kaposi sarcoma, t cell lymphoma, (it includes acute myeloid to chronic or acute leukemia Sample leukaemia, chronic myeloid leukemia, acute lymphatic leukemia, chronic lymphocytic leukemia), childhood One or more in solid tumor, lymphocytic lymphoma；The infectious diseases be selected from HIV, influenza, bleb, Giardiasis, malaria, leishmaniasis, or infected caused by following virus：Hepatitis viruse (such as A type, B-mode or hepatitis C Virus), herpesviral (such as VZV, HSV-1, HAV-6, HSV-II, CMV or angstrom bar Er Shi virus), adenovirus, influenza virus, Vaccinia virus, HTLV viruses, dengue fever virus, papillomavirus, contagiosum, poliovirus, hydrophobin, Huang Virus, echovirus, rhinovirus, Coxsackie virus, coronavirus, Respiratory Syncytial Virus(RSV), mumps virus, rotavirus fiber crops Exanthema virus, rubella virus, parvovirus, JC viruses and arboviral encephalitides virus, or following bacterial infection：Clothing is former Body, rickettsia, pneumococcus, mycobacteria, staphylococcus, streptococcus, meningococcus, conococci, sand Lei Shi It is bacterium, Klebsiella, mycetozoan, pseudomonad, salmonella, comma bacillus, corynebacterium diphtheriae, clostridium botulinum, bacillus anthracis, broken Cold clostridium, Legionella, yersinia pestis, leptospirosis or Lyme disease bacterium, below fungus-caused infection：Aspergillus (such as aspergillus fumigatus, aspergillus niger), Candida (such as Candida albicans), candida krusei, Candida glabrata, Candida tropicalis, Cryptococcus neoformans, Blastomyces dermatitidis, the category (such as mucor, Absidia or rhizopus) of Mucoales, Sporotrichum schenckii, Paracoccidioides brasiliensis, thick ball spore bacterium or blooming histoplasma capsulatum, infection caused by following parasite： Entamoeba historlytica, colon bowel bag worm, good fortune Na Shi worms, spine amoeba, Plasmodium vivax, Babesiamicrofti, suck giardia lamblia, it is hidden Sporozoite, Pneumocystis carinii, T. brucei, Cruz trypanosome, Duo Shi Leishmanias, mouse Earth's bow shock or nippostrongylus brasiliensis； The inflammatory disease be selected from acute diseminated encephalomyelitis, Addision's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, from Body immune hemolytic anemia, oneself immunity hepatitis, arthritis, behcet's disease, american trypanosomiasis, Crohn disease, epidermolysis class Pemphigus, abdominal disease, dermatomyositis, graft versus host disease(GVH disease), Graves disease, type 1 diabetes, Goodpasture syndrome, It is guillain-Barre syndrome, chronic lymphocytic thyroiditis, super IgE syndromes, ITP, lupus erythematosus, multiple hard Change disease, myasthenia gravis, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthrosis Inflammation, xerodermosteosis, temporal arteritis, vasculitis or Wegner's granulomatosis.

Present invention also offers the anti-PD-1 and TEM-8 bispecific antibodies or its variant or its functional fragment Preparation method, it, which is included in, can produce the anti-PD-1 and TEM-8 bispecific antibodies or its variant or its feature piece Host cell of the present invention is cultivated under conditions of section, and anti-PD-1 and TEM-8 bispecific antibodies produced by reclaiming, Or its variant or its functional fragment.

The invention provides can specifically bind the programmed death factor 1 (PD-1) and tumor endothelial mark 8 simultaneously (TEM-8) bispecific antibody or its variant or its functional fragment.The bispecific antibody or its variant of the present invention, Or its functional fragment has at least one of following characteristic：

A. it can be combined with PD-1 with high specific, block PD-1 and PD-L1 interaction；

B. do not combined with other CD28 family members (such as ICOS, CTLA-4 and CD28)；

C. it can be combined with TEM-8 with high specific；

D. do not combined with other VEGFs (such as VEGFR1-3)；

E. immune cell activated (such as tumor specific T cells), and specific recognition TEM-8 positive tumors, promote CD8+ Into solid tumor mass, greatly increase the level of the immunoeffectors such as IFN γ, so as to realize target killing tumor cell.

Present invention also offers humanization PD-1/TEM-8 bispecific antibodies or its variant or its functional fragment.Institute State humanized antibody by be immunized mouse produce mouse source antibody designed via computer simulation and combine display technique of bacteriophage and Obtain, and according to its binding characteristic with PD-1 the and TEM-8 ectodomains of different plant species, identify its corresponding knot Close epitope.The anti-PD-1/TEM-8 bispecific antibodies of humanization of the present invention or its variant or its functional fragment tool The advantageous feature of standby above-mentioned PD-1/TEM-8 bispecific antibodies or its variant or its functional fragment.

Bispecific antibody of the present invention or its variant or its functional fragment can be full length antibodies, or only include Two antigen-binding portion thereofs, such as weight chain variable district (VH), single-stranded (scFv), or full length antibody and merging that antigen-binding portion thereof is constituted Albumen (such as Fig. 2), the model that double Specific binding proteins molecules that any form combination is formed as described above are protected in the present invention In enclosing.

Variant or functional fragment of the present invention include by chemical modification or can increased by mixing in liposome Plus any fragment of half life, fragment reservation and its derived antibodies identical binding specificity and affinity after modification, so The new protein molecular with double specific binding properties obtained is also in the scope of protection of the invention.

On the premise of not substantial effect antibody activity, those skilled in the art can to the present invention sequence replace, Add and/or lack it is one or more (such as 1,2,3,4,5,6,7,8,9 or 10 or more) amino acid, to obtain The variant of the sequence of the antibody or its functional fragment.They are considered as being included in the scope of protection of the invention.

Those skilled in the art can will encode the DNA molecular gram of PD-1/TEM-8 bispecific antibodies of the present invention It is grand into carrier, and then convert host cell.Therefore, present invention also offers by taking bispecific antibody shown in Figure 1A-D as an example Recombinant DNA carrier.The variant of PD-1/TEM-8 bispecific antibodies shown in code pattern 2 and the carrier of functional fragment and host cell Also in the scope of protection of the invention.

Host cell of the present invention can be prokaryotic host cell, eukaryotic host cell or bacteriophage.The protokaryon Host cell can be Escherichia coli, hay bacillus, lactic acid bacteria, streptomycete or proteus mirabilis etc..The eukaryotic host cell Can be such as pichia pastoris phaff, saccharomyces cerevisiae, fission yeast, the insect cell of the fungi of trichoderma, such as meadow mythimna separata, The plant cell of such as tobacco, such as bhk cell, Chinese hamster ovary celI, COS cells, the mammalian cell of myeloma cell.One In a little embodiments, host cell of the present invention is preferably mammalian cell, more preferably bhk cell, Chinese hamster ovary celI, NSO cells or COS cells.

Terms used herein " pharmaceutical composition " represents to combine to realize at least one of certain specific purpose The combination of medicine and optional pharmaceutical acceptable carrier, auxiliary material or therapeutic cells.In certain embodiments, the drug regimen Thing is included in time and/or spatially separated combination, if its can collective effect to realize the purpose of the present invention.For example, Composition contained by described pharmaceutical composition (such as according to the antibody of the present invention, nucleic acid molecules, the combination of nucleic acid molecules and/or is sewed Compound) object, or separate administration can be applied in object with overall.When the composition contained by described pharmaceutical composition dividually When being applied to object, the composition can simultaneously or sequentially be applied to object.Preferably, described pharmaceutical acceptable carrier is water, buffering Solution, isotonic salting liquid such as PBS (phosphate buffer), glucose, mannitol, D-glucose, lactose, starch, stearic acid Magnesium, cellulose, magnesium carbonate, 0.3% glycerine, hyaluronic acid, ethanol, PAG such as polypropylene glycol or triglycerides etc.. Whether the type of pharmaceutical acceptable carrier used is depended particularly on is formulated as being used for oral, nose, intracutaneous, skin according to the composition of the present invention Under, intramuscular or intravenous administration.Addition can be used as comprising wetting agent, emulsifying agent or buffer substances according to the composition of the present invention Agent.It can be applied according to the pharmaceutical composition of the present invention by any suitable approach, such as orally available, nose, intracutaneous, subcutaneous, flesh Interior or intravenous administration.

In a related aspect, combined the invention provides the PD-1/TEM-8 bispecific antibodies with second therapeutic agent Pharmaceutical composition.In one embodiment, the second therapeutic agent be advantageously with PD-1/TEM-8 bispecific antibodies Any agent of combination.The exemplary agents that advantageously can be combined with the PD-1/TEM-8 bispecific antibodies include but not limited In other reagents (including other antibody or its antigen-binding fragment, inhibitor peptides, the small molecular antagonists that suppress PD-1 activity Deng) and/or interference PD-1 upstreams or downstream signal transduction reagent.

The invention provides the side that a kind of PD-1/TEM-8 bispecific antibodies and other treatment method are used in combination Method.In one embodiment, other described therapies are timess advantageously combined with the PD-1/TEM-8 bispecific antibodies Meaning treatment method.The example therapy that advantageously can be combined with the PD-1/TEM-8 bispecific antibodies includes but not limited In with other therapies (including operation, chemotherapy, radiotherapy, cellular immunotherapy etc.) for treating indication of the present invention.

The PD-1/TEM-8 bispecific antibodies that the present invention is provided to PD-1 and TEM-8 while have high-affinity, a side Face be able to can be specifically bound with the PD-1 of activating T cell, the combination of PD-1 acceptors effectively in closing PD-L1 albumen and T cell, And then the Immune escaping mechanism of cancer cell caused by blocking PD-1/PD-L paths, activate T cell tumor killing activity；The opposing party Face can target TEM-8 positive expression tumours, i.e., with specific expressed in tumor vascular endothelium TEM-8 specific bonds, suppress swollen Knurl growth activity and antitumor, realizes the target killing of tumour cell so that the killing tumor effect of the bispecific antibody is bright The aobvious killing tumor effect mediated better than PD-1 and TEM-8 monoclonal antibodies.

Brief description of the drawings

Hereinafter, embodiment of the present invention is described in detail with reference to accompanying drawing, wherein：

Fig. 1 is the structural representation of anti-PD-1/TEM-8 bispecific antibodies of the present invention.

Fig. 2 is the structural representation of the functional fragment or variant of anti-PD-1/TEM-8 bispecific antibodies of the present invention Figure.

Fig. 3 is the SDS-PAGE testing results of purifying humanization PD-1, TEM-8 ectodomain.

Fig. 4 is the ELISA testing results of anti-PD-1 monoclonal antibodies and anti-TEM-8 monoclonal antibodies.

Fig. 5 shows the combination of anti-PD-1 monoclonals 3,21,45 and 77 and CD28 family proteins respectively.

Fig. 6 shows the envelope that anti-PD-1 monoclonals 3,21,45 are combined with 77 pairs of PD-L1 albumen with the PD-1 of human T-cell respectively The effect of closing.

Fig. 7 shows anti-1,5 and 69 specific binding respectively with TEM-8 albumen of TEM-8 monoclonals.

The agarose gel electrophoresis result of the encoding gene of the bispecific antibody of Fig. 8 display present invention.

The recombinant plasmid collection of illustrative plates of the bispecific antibody of Fig. 9 code displayings present invention.

Specific bond of the PD-1/TEM-8 bispecific antibodies of Figure 10 display present invention to PD-1 and TEM-8.

The PD-1/TEM-8 bispecific antibodies of Figure 11 display present invention are respectively to PD-L1 albumen and the PD-1 of people's T cells With reference to sealing process.

The PD-1/TEM-8 bispecific antibodies of Figure 12 display present invention are acted on the Activation In Vitro of human T-cell.

The internal antitumous effect of the PD-1/TEM-8 bispecific antibodies of Figure 13 display present invention.

Figure 14 shows the functional fragment or variant of PD-1/TEM-8 bispecific antibodies of the present invention shown in Fig. 2 Internal antitumous effect.

Embodiment

Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only For illustrating the present invention, it does not limit the scope of the present invention in any way.

Experimental method in following embodiments, is conventional method unless otherwise specified.Original used in following embodiments Material, reagent material etc., unless otherwise specified, are commercially available products.

The structure design of the PD-1/TEM-8 bispecific antibodies of embodiment 1

PD-1 and the bispecific antibody of TEM-8 ectodomains can be specifically bound simultaneously the invention provides some Structural model, the structure of this 4 kinds of principle example of antibody-like as shown in figure 1, in Figure 1A-C the formation of 3 kinds of heterodimers be based on CH3 domains mutating technology (knob and hole mutation) (Von known to art technology person Kreudenstein et al., MAbs.2013 5 (5):646-54).Wherein, the light chain of (A) anti-PD-1 specific antibodies is (anti- PD-1 VL-CL)-heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) pair and anti-TEM-8 specific antibodies light chain it is (anti- PD-1 VL-CL)-heavy chain (anti-PD-1 VH-CH1- hinge areas-CH2-CH3) is to the bispecific binding protein of composition；(B) resist The light-heavy chain pair of PD-1 specific antibodies and anti-TEM-8 light chain variable districts (anti-TEM-8 VL), weight chain variable district (anti-TEM- 8 VH), hinge area and Fc fragments (anti-TEM-8 CH2-CH3) fusion protein composition bispecific binding protein；(C) anti-PD-1 The light-heavy chain pair of light chain variable district, weight chain variable district, hinge area and Fc fragment fusion proteins and anti-TEM-8 specific antibodies The bispecific binding protein of composition；(D) heavy chain of the light chain of anti-PD-1 specific antibodies and anti-PD-1 specific antibodies, with And the bispecific of anti-TEM-8 light chain variable districts (anti-TEM-8 VL) and weight chain variable district (anti-TEM-8 VH) fusion protein composition Associated proteins.

The variant of PD-1/TEM-8 bispecific antibodies and the embodiment of functional fragment are as shown in Fig. 2 in Fig. 2 E-H The formation of bispecific antibody variant and functional fragment based on known to art technology person fusogenic peptide technology (with polypeptide chain or Protein connects different structure territory, and then realizes amalgamation and expression), the formation of heterodimer is based on art technology in Fig. 2 I The weight chain variable district (anti-PD-1 VH) of CH3 domains mutating technology (same to Figure 1A-C) (E) anti-PD-1 known to member is with resisting The fusion protein of TEM-8 weight chain variable districts (anti-TEM-8 VH)；(F) anti-PD-1 single chain variable fragment (scFv) and anti-TEM-8 ScFv composition fusion protein；(G) non-the exempting from of anti-PD-1 single chain variable fragment (scFv) and anti-TEM-8 scFv compositions Epidemic disease Immunoglobulin fusion albumen；(H) anti-PD-1 light chain variable districts, weight chain variable district, hinge area, Fc fragments can with anti-TEM-8 light chains Become area, the tetravalent fusion protein of weight chain variable district composition；(I) anti-PD-1 light chain variable districts, weight chain variable district, hinge area and Fc Fragment fusion protein is double special with anti-TEM-8 light chain variable districts, weight chain variable district, hinge area and Fc fragment fusion proteins composition Property associated proteins (note：Specific recognition PD-1 and TEM-8 function fragment position are interchangeable in figure).

The system of the anti-PD-1/TEM-8 bispecific antibodies antigen of embodiment 2 (people PD-1 and TEM-8 extracellular can solubilization domain) It is standby

The coding gene sequence of people source PD-1 and TEM-8 ectodomain is obtained in UniProt databases, passes through full genome The mode of synthesis obtains encoding gene PD-1ED-CDS and TEM-8ED-CDS, and synthetic product is through BamH I, EcoR I (PD- 1ED-CDS) and after BamH I, Xba I (TEM-8ED-CDS) double digestion, pcDNA3.1 carriers are cloned into, 293T cells are utilized (ATCC) protein expression is carried out as host cell, using purification techniques such as affinity chromatography, ion-exchange chromatographies to purpose egg Purified in vain.

Fig. 3 behaviours source PD-1 and TEM-8 ectodomains protein purification simultaneously carry out the SDS-PAGE after deglycosylation processing Testing result figure.

The anti-human PD-1 of embodiment 3 and TEM-8 antibody acquisition

1. immune animal

The purified people PD-1 of 1mg/ml or the μ l of TEM-8 ectodomains protein 10 0 is complete with isometric Freund respectively After adjuvant is uniformly mixed at room temperature, in subcutaneous abdomen multi-point injection to 5 6-8 weeks BALB/C mice bodies.After 7 days, by 1mg/ After the μ l of ml protein 10s 0 are uniformly mixed at room temperature with isometric incomplete Freund's adjuvant, subcutaneous abdomen multi-point injection to Mice Body It is interior.After seven days, repeat the above steps.After seven days, repeat the above steps.Three days after 4th injection, disconnected rat-tail point takes the μ l of blood 15, 2h is stored at room temperature, with 25 DEG C, 6000-8000rpm centrifugation 10-15min, supernatant is taken, passes through ELISA experiment detection potency.If effect The available i.e. eye of valency detection takes whole blood, is stored at room temperature 2h, with 25 DEG C, 6000-8000 rpm centrifugation 10min, takes supernatant, -80 DEG C Preserve.If potency is unavailable, continue to be immunized once.

2. cell fusion

It is sterile to take mouse boosting cell and cell suspension is made, by 1 × 10⁸Individual splenocyte and 2-5 × 10⁷Individual myeloma (SP2/ 0-Ag14) cell fusion, fusion agent is made with 50%PEG, after HAT Selective agar mediums culture 3-10 days, is carried out with HT culture mediums Culture, condition of culture is 5%CO₂, 37 DEG C.

3. positive colony is screened

PD-1 or TEM-8 ectodomain eggs are combined using that can be secreted in ELISA reaction screening monoclonal hybridomas Bai Kangti clone, wherein with reference to PD-1 4 plants of preferred clone (clone 3,21,45,77), with reference to TEM-8 preferred clone 3 Strain (clone 1,5,69), the ELISA testing results of above clones secrete antibody as illustrated in figures 4 a andb, the clone filtered out The corresponding antigen protein of specific recognition.

The identification that the anti-PD-1 antibody of embodiment 4 is combined with other CD28 family proteins

The specificity of the PD-1 antibody gone out for evaluation and screening, by member protein PD-1, CD28, CTLA-4 of CD28 families Coating 2h is stood in 37 DEG C of incubators in ELISA Plate with ICOS (R＆D Systerm)；4 DEG C of closings are stayed overnight；Added after washing 100 μ l candidates antibody (1 μ g/ml), are incubated 1h at 37 DEG C；The goat anti-mouse antibody of the HRP marks of dilution is added after washing 5 times (Abcam), it is incubated at room temperature 1h；The 2M that 50 μ l/ holes are added after the μ l/ holes of OPD substrate solutions 100, colour developing 5min is added after washing 5 times H₂SO₄Terminating reaction.The light absorption value in each hole under 490nm is determined with ELIASA.Experimental result such as Fig. 5, the antibody of candidate clone secretion CD28 family member's albumen in addition to PD-1 is not combined.

The enclosed experiment that the PD-1 acceptors of the anti-PD-1 antibody on human T cell of embodiment 5 are combined with PD-L1

1. the separation of human peripheral T cell

Healthy volunteer peripheral blood 10mL is extracted, isometric PBS is added into pipe, cell suspension 20mL is gently blown and beaten into, Cell suspension is slowly added on to the top layer of 20mL LTS1077 lymphocytes separating solutions with suction pipe, with 2000 revs/min of centrifugations 20min, sucks the blood plasma of the superiors, and the monocyte drawn under plasma layer is inserted in centrifuge tube, is washed 3 times with PBS, obtains Human peripheral blood mononuclear cell PBMC.

Suspension is made with PBS in isolated PBMC, adds 100 μ l/ml CD3 antibody (Invitrogen), Featheriness is mixed, and is incubated at room temperature 15min；Add and 60min, every 10 points are incubated at 5 μ l/ml immunomagnetic beadses (Invitrogen), 4 DEG C Clock shakes once；Rinsed and diluted with PBS, cell suspension injection is filled with the special test tube of stainless steel wool, separator is put into Isolated and purified, T cell can not be purified by separator under high-intensity magnetic field, Fig. 6 A are the mirror for the T cell purity isolated Determine result.

2. the preparation of human PD-L 1 ectodomain and green fluorescent protein fusion protein

The coding gene sequence of human PD-L 1 ectodomain, the side synthesized by full genome are obtained in UniProt databases Formula obtains encoding gene, and synthetic product is cloned into pEGFP-N1 carriers after EcoR I, Xho I double digestions, utilizes 293T cells (ATCC) carry out protein expression as host cell, utilize the purification techniques such as affinity chromatography, ion-exchange chromatography Destination protein is purified, the people source PD-L1 ectodomain albumen rh-PD-L1- of green fluorescent protein amalgamation and expression is obtained GFP, Fig. 6 B are the expression and purification of fusion protein and carry out the SDS-PAGE results after deglycosylation processing.

3. the enclosed experiment that the PD-1 acceptors of anti-PD-1 antibody on human T cell are combined with PD-L1

The antibody (1 μ g/ml) that preferably 4 strain clones are produced softly is mixed at room temperature with rh-PD-L1-GFP (1 μ g/ml) 30min, then adds mixed protein solution the human peripheral T cell isolated and purified out, clear with PBS after being incubated at room temperature 15 minutes Wash 3 times, analyzed with flow cytometer.

Fig. 6 C-G displays clone 3,21 and 77 can hinder the combination of the PD-1 acceptors on PD-L1 and human T-cell, and clone 45 Barrier effect it is poor.

The identification of the anti-TEM-8 antibody of embodiment 6 and EFGR, HER2 and VEGFR combination

TEM-8, EFGR, HER2 and VEGFR (R＆D Systerm) are stood into coating in ELISA Plate in 37 DEG C of incubators 2h；4 DEG C of closings are stayed overnight；Added after washing at 100 μ l candidates antibody (1 μ g/ml), 37 DEG C and be incubated 1h；Added after washing 5 times dilute The HRP mark goat anti-mouse antibodies (Abcam) released, are incubated at room temperature 1h；The μ l/ holes of OPD substrate solutions 100 are added after washing 5 times, The 2M H in 50 μ l/ holes are added after colour developing 5min₂SO₄Terminating reaction.The light absorption value in each hole under 490nm is determined with ELIASA.Experiment As a result as shown in fig. 7, the antibody of candidate clone secretion does not combine EFGR, HER2 and VEGFR albumen.

The acquisition of the variable region sequences of the anti-PD-1 and TEM-8 candidate antibodies of embodiment 7

Expand the anti-PD-1 of culture respectively and clone 3,21,77 and anti-TEM-8 clones 1,5,69, culture to cell concentration is 10⁹It is individual, Cell is collected by centrifugation, removes cell culture fluid, cell is gently washed twice with the PBS (6-8mL) of precooling；With Trizol kits (TAKARA) extract total serum IgE, with UV spectrophotometer measuring RNA purity and concentration；Using total serum IgE as mould Plate, reverse transcription obtains cDNA, and reverse transcription system is 20 μ l, wherein 5 × reverse transcription Buffer (4 μ l), dNTP (1 μ l), MgCl₂ (2.4 μ l), PD (N6) random primer (1.5 μ l), RNA templates (2 μ g), reverse transcriptase (1 μ l), DEPC water (to 20 μ l).

Using cDNA as template, the DNA sequence dna of variable region in amplified hybridization knurl, amplimer sequence and antibody variable region first Framework region and constant region complementation (Orlandi R, et al., Cloning immunoglobulin variable domains for expression by polymerase chain reaction.Proc.Natl.Acad. Sci.USA,1989,86： 3833), amplification system is 50 μ l, wherein 5 × PCR Buffer (10 μ l), dNTP (4 μ l), primer (2 μ l), cDNA templates (1 μ L), PCR enzymes (0.5 μ l), PCR water (to 50 μ l).

Amplified production is cloned into pMD19 carriers, and recon sequencing is carried out after being screened through blue hickie, obtains anti-PD-1 clones 3rd, 21,77 heavy chain and the amino acid sequence of light chain variable district are respectively SEQ ID No:1 and 3,5 and 7,9 and 11；Encode phase The nucleotide sequence for answering amino acid is respectively SEQ ID No:2 and 4,6 and 8,10 and 12.Obtain anti-TEM-8 and clone 1,5,69 Heavy chain and the amino acid sequence of light chain variable district are respectively SEQ ID No:13 and 15,17 and 19,21 and 23；Encode corresponding amino The nucleotide sequence of acid is respectively SEQ ID No:14 and 16,18 and 20,22 and 24.

The anti-PD-1 and TEM-8 antibody of embodiment 8 it is humanization modified

Utilized according to the variable region amino acid sequence (sequence that embodiment 7 is obtained) of the anti-PD-1 of acquisition and TEM-8 antibody IMGT/V-Quest line servers analyze antibody subtype, the CDR region of tri- kinds of antibody of comprehensive Kabat, Chothia and IMGT Numbering schemes determine six CDR region sequences of antibody light chain and heavy chain；Mouse parental antibody Fab is carried out using DS softwares And its chimeric humanized antibody Fab space structure, obtain chimeric antibody molecular model after, to parent Fab and chimeric antibody Fab initial models are comprehensively optimized using molecular dynamics simulation, extract stable average conformation；Examined using epitope scanning The humanized antibody linear epitope that may be present and comformational epitope of design are tested, it is determined that the Framework Region amino acid site that must retain Afterwards, different amino acid mutation sites are designed and obtains humanized antibody.

On this basis, we have obtained multiple humanized antibodies, wherein scoring highest anti-PD-1 and anti-TEM-8 resist Body sequence is respectively clone 81 and clone 76, and the heavy chain and light-chain amino acid sequence of clone 81 are respectively SEQ ID No:25 Hes 27, the nucleotide sequence for encoding corresponding amino acid is respectively SEQ ID No:26 and 28；The heavy chain and light chain amino acid of clone 76 Sequence is respectively SEQ ID No:29 and 31, the nucleotide sequence for encoding corresponding amino acid is respectively SEQ ID No:30 and 32.

The structure of the anti-PD-1/TEM-8 bispecific antibodies expression vector of embodiment 9

The anti-PD-1/TEM-8 bispecific antibodies moderate resistance PD-1's (clone 81, preparation method is shown in embodiment 8) of the present invention Variable region coding nucleotide is PD-1-VH and PD-1-VL, the variable region of anti-TEM-8 (clone 76, preparation method is shown in embodiment 8) Coding nucleotide is TEM-8-VH and TEM-8-VL, IgG1 heavy chain constant region CH1, hinge area, Fc and Fc-knob and Fc-hole Coding nucleotide, and constant region of light chain CL kappa coding nucleotide, and link scFv polypeptide (GGGGS) 3 encode Gene is obtained from Life Technologies Inc. (Carlsbad, CA).

Each different chain shown in Fig. 1 in the bispecific antibody of 4 kinds of structures, VL and CL, VH and CH1, CH1 and Fc (Fc-knob, Fc-hole), scFv and Fc (Fc-knob, Fc-hole), Fc and scFv are obtained by way of over-lap PCR. PcDNA3.1 and pIRES as expression vector to prepare bispecific antibody, according to vector multiple cloning site and encoding gene sequence Row design primer (being shown in Table 1).

The PCR primer sequence of table 1

PD-1-VH, PD-1-VL, TEM-8-VH, TEM-8-VL, CH1, hinge area (Hinge), Fc, Fc-knob, Fc- Hole and CL amplification system is 50 μ l, wherein 5 × PCR Buffer (10 μ l), dNTP (4 μ l), primer (2 μ l), cDNA moulds Plate (1 μ l), PCR enzymes (0.5 μ l), PCR water (to 50 μ l).Amplification condition is：98 DEG C are incubated 3 minutes, then 25 circulations：98 DEG C denaturation 30 seconds, 60 DEG C anneal 30 seconds, 72 DEG C extend 1 minute；Extended 10 minutes with 72 DEG C of closures.Fig. 8 A are the fine jade of PCR primer Lipolysaccharide electrophoresis result figure.

Using over-lap PCR by PD-1-VL and CL；PD-1-VH, CH1, hinge area (Hinge) and Fc-knob/Fc-hole； TEM-8-VL and CL；TEM-8-VH, CH1, hinge area and Fc-hole/Fc-knob；PD-1 scFv(PD-1-VL、(GGGGS) 3rd, PD-1-VH), hinge area (Hinge) and Fc-knob/Fc-hole；TEM-8 scFv(TEM-8-VL、(GGGGS)3、TEM- 8-VH), hinge area and Fc-hole/Fc-knob；PD-1-VH, CH1, hinge area (Hinge), Fc and TEM-8 scFv are linked into The encoding gene of each antibody chain shown in Fig. 1, the μ l of PCR system 50, wherein 5 × PCR Buffer (10 μ l), dNTP (4 μ l), draw Thing (2 μ l), cDNA templates (each 1 μ l of PCR primer), PCR enzymes (0.5 μ l), PCR water (to 50 μ l) amplification condition is：98 DEG C of incubations 3 minutes, then 30 circulations：98 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute；Extend 10 with 72 DEG C of closures Minute.Fig. 8 B are the agarose electrophoresis result figure of PCR primer.Each chain coding sequence is SEQ ID No: 34、36、38、40、 42、44、46.It will be built after purpose fragment digestion to expression vector pcDNA3.1, the restructuring of 4 kinds of bispecific antibodies shown in Fig. 1 Plasmid map is shown in Fig. 9 A-G, and (anti-PD-1 is light for coding PD-1-VL and CL by wherein Fig. 9 A recombinant plasmids pcDNA3.1-PD-1-LC Chain) collection of illustrative plates；Fig. 9 B recombinant plasmids pcDNA3.1-TEM-8-LC is coding TEM-8-VL and CL (anti-TEM-8 light chains) collection of illustrative plates；Fig. 9 C Recombinant plasmid pcDNA3.1-PD-1-HC is (anti-for coding PD-1-VH, CH1, hinge area (Hinge) and Fc-knob/Fc-hole PD-1 heavy chains) collection of illustrative plates；Fig. 9 D recombinant plasmids pcDNA3.1-TEM-8-HC is coding TEM-8-VH, CH1, hinge area (Hinge) With Fc-knob/Fc-hole (anti-TEM-8 heavy chains) collection of illustrative plates；Fig. 9 E recombinant plasmid pcDNA3.1-PD-1 scFv-Fc are coding PD- 1 scFv and Fc-knob/Fc-hole collection of illustrative plates；Fig. 9 F recombinant plasmid pcDNA3.1-TEM-8 scFv-Fc are coding TEM-8 ScFv and Fc-knob/Fc-hole collection of illustrative plates；Fig. 9 G recombinant plasmid pcDNA3.1-PD-1-HC-TEM-8 scFv are coding PD-1- VH, CH1, hinge area (Hinge), Fc and TEM-8 scFv collection of illustrative plates.

The preparation of the PD-1/TEM-8 bispecific antibodies of embodiment 10

1. utilize 293F host cell expression bispecific antibodies

24 hours before transfection, by 1 × 10⁶Individual 293F cells are inoculated in the 293freestyle cultures in flask In base, 37 DEG C, 8%CO₂, cultivate under the conditions of 130rpm.100 μ l 293fectin are added in 1ml OPtiMEM, stirring bar Under part, it is incubated 5 minutes at room temperature.Meanwhile, recombinant plasmid is combined as shown in table 2, is dissolved in 1ml OPtiMEM, DNA is total Measure as 30 μ g.Above-mentioned DNA and 293fectin is thoroughly mixed, and cumulative volume is 2ml, in incubation at room temperature 20 minutes.Then Add the mixture into cell culture.Being cultivated at 37 DEG C in cell, and incubator has 5%CO₂, trained with 130rpm Support 5 days.

Each carrier component of the rotaring redyeing system of table 2

Antibody is numbered	Carrier combinations
		Figure 1A	PD-1-LC：PD-1-HC：TEM-8-HC：TEM-8-LC=1:2:2:1
Figure 1B	PD-1-LC：PD-1-HC：TEM-8-scFv-Fc=1:2:3
		Fig. 1 C	PD-1-scFv-Fc：TEM-8-VH：TEM-8-VL=3:2:1
Fig. 1 D	PD-1-LCD：PD-1-HC-TEM-8-scFv=1:3

2. antibody purification

Cell culture is centrifuged based on 2000 × g, supernatant is collected and is filtered with 0.22 μm of filter.The liquid of collection is used 10 times of (being calculated with volume) combination buffer (9.5mM NaH₂PO₄+40.5mM Na₂HPO₄, pH 7.0) and dilution, then pass through Sepharose Fast Flow protein A affinity chromatographic columns (being purchased from GE companies, 5ml volumes), FabAffinity KBPAgarose is affine filler (is purchased from ACROBiosystems companies, 5ml volumes), and SP cation-exchange chromatography posts (are purchased from GE companies, 10ml) purified, operated according to service manual.

The identification of the PD-1/TEM-8 bispecific antibodies of embodiment 11 specific binding

PD-1, CTLA-4, TEM-8 and VEGFR (R＆D Systerm) are stood into coating in ELISA Plate in 37 DEG C of incubators 2h；4 DEG C of closings are stayed overnight；4 kinds of antibody (, referring to embodiment 8, structure is referring to Figure 1A-D for sequence) (100 μ g/ after purification are added after washing Ml gradient dilution liquid (PBS serial dilutions (1)：102,1：103,1：104,1：105,1：106,1：107,1：108,1： 109), 100 μ l/ holes, are incubated 1h at 37 DEG C；The HRP mark goat anti-mouse antibodies (Abcam) of dilution are added after washing 5 times, It is incubated at room temperature 1h；The 2M H that 50 μ l/ holes are added after the μ l/ holes of OPD substrate solutions 100, colour developing 5min are added after washing 5 times₂SO₄Eventually Only react.The light absorption value in each hole under 490nm is determined with ELIASA.As shown in Figure 10,4 kinds of bispecific antibodies can be same for experimental result When specific recognition PD-1 and TEM-8.

The closing that the anti-PD-1/TEM-8 bispecific antibodies of embodiment 12 are combined to the PD-1 acceptors of human T-cell with PD-L1 Experiment

By 4 kinds of bispecific antibodies (, referring to embodiment 8, structure is referring to Figure 1A-D for sequence) (1 μ g/ml) and rh-PD-L1- GFP (1 μ g/ml) is soft at room temperature to mix 30min, and then mixed protein solution added to the human peripheral T isolated and purified out With PBS 3 times after cell, incubation at room temperature 15 minutes, analyzed with flow cytometer.As shown in figure 11,4 kinds of bispecifics Antibody can hinder the combination of the PD-1 acceptors on PD-L1 and human T-cell.

The experiment of the PD-1/TEM-8 bispecific antibody Activation In Vitro T cells of embodiment 13

By the fresh PBMC bed boards for preparing human peripheral into 96 hole flat undersides, after overnight incubation, add 10ug/ml's Antibody and 100ng/ml tetanus toxin (TT), culture collect supernatant after 3 days, with Life Technology companies Luminex instrument and the CD8+ cytokine detection kits detection bispecific antibody and the IL2 secretion levels of control group of EMD companies (result is shown in Figure 12).As a result show, the humanized antibody can stimulate the function of T cell, the PD- of 4 shown in Fig. a kind form 1/TEM-8 bispecific antibodies (sequence is referring to embodiment 8) can effectively activate T cell function, and wherein Fig. 1 D-shapeds formula is double special Xenoantibody activation effect is relatively preferable, and IL-2 secretes caused by the PD-1/TEM-8 bispecific antibodies of control group and 4 kinds of forms The gaining rate of amount is respectively 115.2%, 291.5%, 278.2%, 322.2% and 341.7%.

Table 3 activates the experiment of T cell function

Note：Compared * * P with control group<0.01.

The in-vivo tumour killing experiments of the anti-PD-1/TEM-8 bispecific antibodies of embodiment 14

1. different plant knurl model is set up

CT-26 cells in exponential phase are resuspended in the RPMI 1640 culture mediums of serum-free so that cell concentration For 1 × 10⁷Individual cell/ml.By cell infusion in the left shoulder back of mouse, every 200 μ l are designated as experiment 0 day.Survey one within every two days The longest diameter of secondary lotus knurl and most short diameter, the volume formula of lotus knurl：Volume (mm³)=long × wide²/2.Test the 30th day, swell The volume of knurl has reached 500mm³, mouse is put to death, tumour is separated.

2. anti-PD-1/TEM-8 bispecific antibodies kill tumor experiment

Plantation knurl mouse is divided into 4 groups, every group 4, with the PD-1/TEM-8 bispecific antibodies of purifying (sequence referring to Embodiment 8, as shown in Fig. 1 D) tail vein injection is into knurl Mice Body, and consumption is respectively A groups：0mg/kg, B group：5mg/ Kg, C group：50mg/kg and D groups：100mg/kg.Inject 1 time week about, it is continuous to inject 4 times, record mouse interior tumor size Change.PD-1/TEM-8 bispecific antibodies not only suppress the growth of mouse interior tumor, while also having the work for reducing tumour With after being injected 6 weeks with 50mg/kg and 500mg/kg dosage, gross tumor volume is contracted to 54.7% He of initial volume respectively 41.7% (see Figure 13 A).

The dual anti-inhibitory action result to tumour of table 4

Group	Tumor size	Gross tumor volume gaining rate
			Control group	1120±54mg	1178.9%
5mg/kg groups	389±40mg**	413.8%
			50mg/kg groups	52±20mg**	54.7%
500mg/kg groups	40±22mg**	41.7%

Note：Compared * * P with control group<0.01.

3.PD-1 monoclonal antibodies, TEM-8 monoclonal antibodies are imitated with anti-PD-1/TEM-8 bispecific antibodies tumor-killing Rate comparative experiments

Plantation knurl mouse is divided into 4 groups, every group 4, with the PD-1 monoclonal antibodies (clone prepared by embodiment 8 of purifying 81), TEM-8 monoclonal antibodies (embodiment 8 prepare clone 76) and anti-PD-1/TEM-8 bispecific antibodies (sequence referring to It is embodiment 8, dual anti-shown in Fig. 1 D) tail vein injection is into knurl Mice Body, and consumption is respectively A groups：PBS, B group：50mg/kg PD-1 monoclonal antibodies (clone 81), C groups：50mg/kg TEM-8 monoclonal antibodies (clone 76) and D groups：The anti-PD-1/TEM-8 of 50mg/kg are double special Heterogenetic antibody (dual anti-shown in Fig. 1 D).Inject 1 time week about, continuous to inject 4 times, record mouse interior tumor size becomes Change.The effect that anti-PD-1/TEM-8 bispecific antibodies reduce tumour is substantially better than PD-1 monoclonal antibodies and TEM-8 monoclonal antibodies, mono- in PD-1 In the presence of anti-, TEM-8 monoclonal antibodies and anti-PD-1/TEM-8 are dual anti-, gross tumor volume is contracted to the 64.9% of initial volume respectively, 81.8% and 51.1% (see Figure 13 B, p ＜ 0.01).

Inhibitory action result of the different antibodies of table 5 to tumour

Group	Tumor size	Gross tumor volume gaining rate
			Control group	1239±49mg	1264.3%
PD-1 monoclonal antibody groups	61±22mg**	64.9%
			TEM-8 monoclonal antibody groups	81±25mg**	81.8%
Anti- dual anti-group of PD-1/TEM-8	48±20mg**#&	51.1%

Note：Compared * * P with control group<0.01.Compared with PD-1 monoclonal antibody groups^#P<0.05.Compared with TEM-8 monoclonal antibody groups^&P< 0.05。

Variant (5 kinds of variants shown in Fig. 2) in-vivo tumour killing experiments of the anti-PD-1/TEM-8 bispecific antibodies of embodiment 15

Plantation knurl mouse is divided into 6 groups, every group 4, with the variant of 5 kinds of purifying anti-PD-1/TEM-8 bispecific antibodies (the dual anti-variant shown in Fig. 2 E-I, sequence referring to clone 81 and clone 76, preparation method is shown in embodiment 8) tail vein injection into In knurl Mice Body, consumption is respectively A groups：PBS, B group：50mg/kg variants 1 (Fig. 2 E), C groups：50mg/kg variants 2 (Fig. 2 F), D Group：50mg/kg variants 3 (Fig. 2 G), E groups：50mg/kg variants 4 (Fig. 2 H), and F groups：50mg/kg variants 5 (Fig. 2 I).Every one Week injection 1 time, it is continuous to inject 4 times, record mouse interior tumor size variation.5 kinds of anti-PD-1/TEM-8 bispecific antibodies Variant is likewise supplied with suppressing effect of tumour growth, and in the presence of variant 1-5, gross tumor volume is developed to initial volume respectively 100.5%, 71.1%, 72.1%, 69.6%, 64.2% and 65.5% (see Figure 14, p ＜ 0.01).

Inhibitory action result of the variant of the different antibodies of table 6 to tumour

Note：Compared * * P with control group<0.01.

<110>Along sky cellular biological technique（Tianjin）Limited company

<120>Anti- PD-1 and TEM-8 bispecific antibodies and its application

<130> SHUNHOBiMab-PD-1XTEM-8

<160> 46

<170> PatentIn version 3.3

<210> 1

<211> 120

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 3 heavy chains

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gly Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Asn Glu Lys Phe

50 55 60

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 2

<211> 360

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 3 weight chain variable districts

<400> 2

cagggccagc tggtgcagag cggcggcgag gtgaagaagc ccggcgccag cctgaggctg 60

gactgcaagg ccagcggcta caccttcacc aactactaca tgcactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atctggtacg acggcagcaa gaggtactac 180

aacgagaagt tcaagaacag ggtgaccatc accgccgaca agagcaccag caccgcctac 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggagggac 300

tacttcttcg actggtactt cgactactgg ggccagggca ccaccgtgac cgtgagcagc 360

<210> 3

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 3 light chain variable districts

<400> 3

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

1 5 10 15

Glu Arg Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser

20 25 30

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

35 40 45

Arg Leu Leu Ile Tyr Asp Ala Ser Tyr Leu Glu Ser Gly Ile Pro Ala

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Ser

85 90 95

Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 4

<211> 333

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 3 light chain variable districts

<400> 4

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccagc 60

atcagctgca gggccagcca gagcgtgagc accagcggct acagctacct ggcctggtac 120

cagcagaagc ccggccaggc ccccaggctg ctgatctacg acgccagcta cctggagagc 180

ggcatccccg ccaggttcag cggcagcggc agcggcaccg acttcaccct gaagatcagc 240

agcctggagc ccgaggacgt gggcgtgtac tactgccagc acagcagcaa ctggcccctg 300

accttcggcc agggcaccaa gctggagatc aag 333

<210> 5

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 21 weight chain variable districts

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Gly Tyr Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys

50 55 60

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

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr

85 90 95

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

100 105 110

Ser Ser

<210> 6

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 21 weight chain variable districts

<400> 6

caggtgcagc tggtggagag cggcgccgag gtggtgcagc ccggcaggag cctgaggctg 60

gactgcaagg ccagcggcta catcaccttc agcgactact acatgtactg ggtgaggcag 120

gcccccggcc acggcctgga gtggatcggc tacatcaacc ccagcaacgg cggcaccaac 180

ttcaacgaga agttcaaggg caggttcacc atcagcaggg acaacagcaa gaacaccctg 240

ttcctgcaga tgaacagcct gcagttcgac gacaccgccg tgtactactg cgccaccaac 300

gacgactact ggggccaggg caccctggtg accgtgagca gc 342

<210> 7

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 21 light chain variable districts

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Arg Asp Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 21 light chain variable districts

<400> 8

gacatcgtga tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccagc 60

atcagctgca gggccagcaa gggcgtgagc agctacctgc actggtacca gcagaagccc 120

ggccagagcc ccaggctgct gatctacctg gccagctacc tggagagcgg cgtgcccgac 180

aggttcagcg gcagcggcag cggcaccgac ttcaccctga agatcagcag ggtggaggcc 240

gaggacttcg ccgtgtacta ctgccagcag agcagggacc tgccctacac cttcggccag 300

ggcaccaagc tggagatcaa g 321

<210> 9

<211> 115

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 77 weight chain variable districts

<400> 9

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

1 5 10 15

Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ser Trp Asn

20 25 30

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

35 40 45

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

50 55 60

Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 10

<211> 345

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 77 weight chain variable districts

<400> 10

cagggccagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccac ctgcaccgtg 60

accggctaca gcatcaccag cgactacagc tggaactgga tcaggcaggc ccccatccac 120

ggcctggagt ggatcggcta catcaactac gccggcagca ccagctacaa ccccagcctg 180

gaggccgtga ccatcaccgc cgacaagagc accagcaccg cctacatgga gctgagcagc 240

ctgaggagcg aggacaccgc cgtgtactac tgcgccaggt ggttcggcag caccgcctgg 300

tacatcgacg tgtggggcca gggcaccacc gtgaccgtga gcagc 345

<210> 11

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 77 light chain variable districts

<400> 11

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gln

1 5 10 15

Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ala Leu Leu His Ser Asp

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Glu Leu Ser Ser Arg Phe Ser Gly Ile Pro Asp

50 55 60

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

65 70 75 80

Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly Val His Ile

85 90 95

Pro Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 327

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 77 light chain variable districts

<400> 12

gacgtggtga tgacccagag ccccctgagc ctgcccgtga ccctgcagcc cgccagcatc 60

agctgcaaga gcagccaggc cctgctgcac agcgacggca agacctacct gtactggtac 120

ctgcagaagc ccggccagag ccccaagctg ctgatctacg agctgagcag caggttcagc 180

ggcatccccg acaggatcac cggcagcggc accgacttca ccctgaagat cagcagggtg 240

gaggccgagg acctgggcgt gtacttctgc ttccagggcg tgcacatccc ctacagcttc 300

ggccagggca ccaagctgga gatcaag 327

<210> 13

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 1 weight chain variable district

<400> 13

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 Gly Phe Ser Phe Asn Ala Tyr Ala

20 25 30

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

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Ala Gly Phe His Pro Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 14

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 1 weight chain variable district

<400> 14

caggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccggcttcag cttcaacgcc tacgccatga gctgggtgag gcaggccccc 120

ggcaagggcc tggagtgggt gagcctgatc agcaccggca gcgccaccta ctacgccgac 180

agcctgaagg gcaggttcac catcagcagg gacaacagca agaacaccct gtacctgcag 240

atgaacagcc tgagggccga ggacaccgcc gtgtactact gcgccagggc cggcttccac 300

cccgacaact ggggccaggg caccctggtg accgtgagca gc 342

<210> 15

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 1 light chain variable district

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asp Ser Asn Arg Pro Ser Gly Tyr Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

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

85 90 95

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 16

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 1 light chain variable district

<400> 16

gacatcgagc tgacccagcc ccccagcgtg agcgtggccc ccggccagac cgccaggatc 60

agctgcagcg gcgacagcat ccccaactac agcgtgagct ggtaccagca gaagcccggc 120

caggcccccg tgctggtgat ctacgccgac agcaacaggc ccagcggcta ccccgagagg 180

ttcagcggca gcaacagcgg caacaccgcc accctgacca tcagcggcac ccaggccgag 240

gacgaggccg actactactg ccagagctac gacaacacca gccccgacct gttcggcggc 300

ggcaccaagc tgaccgtgct g 321

<210> 17

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 5 weight chain variable districts

<400> 17

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 Gly Phe Ala Phe Asn Ser Tyr Ala

20 25 30

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

35 40 45

Leu Ile Ala Ser Gly Ser Ala Thr Tyr Tyr Ala Asp Ser Ile His Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Ala Gly Phe Lys Pro Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 18

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 5 weight chain variable districts

<400> 18

gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccggcttcgc cttcaacagc tacgccatga gctgggtgag gcaggccccc 120

ggcaagggcc tggagtgggt gagcctgatc gccagcggca gcgccaccta ctacgccgac 180

agcatccacg gcaggttcac catcagcagg gacaacagca agaacaccct gtacctgcag 240

atgaacagcc tgagggccga ggacaccgcc gtgtactact gcgccagggc cggcttcaag 300

cccgacaact ggggccaggg caccctggtg accgtgagca gc 342

<210> 19

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 5 light chain variable districts

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asp Ser Asn Arg Pro Ser Gly Tyr Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

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

85 90 95

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 20

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 5 light chain variable districts

<400> 20

gacatcgagc tgacccagcc ccccagcgtg agcgtggccc ccggccagac cgccaggatc 60

agctgcagcg gcgacagcat ccccaactac agcgtgagct ggtaccagca gaagcccggc 120

caggcccccg tgctggtgat ctacgccgac agcaacaggc ccagcggcta ccccgagagg 180

ttcagcggca gcaacagcgg caacaccgcc accctgacca tcagcggcac ccaggccgag 240

gacgaggccg actactactg ccagagctac gacaacacca gccccgacct gttcggcggc 300

ggcaccaagc tgaccgtgct g 321

<210> 21

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 69 weight chain variable districts

<400> 21

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 Gly Phe Thr Phe Asn Ser Tyr Ala

20 25 30

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

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Ala Gly Phe His Pro Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 22

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 69 weight chain variable districts

<400> 22

caggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccggcttcac cttcaacagc tacgccatga gctgggtgag gcaggccccc 120

ggcaagggcc tggagtgggt gagcctgatc gccagcggca gcgccaccta ctacgccgac 180

agcgtgaagg gcaggttcac catcagcagg gacaacagca agaacaccct gtacctgcag 240

atgaacagcc tgagggccga ggacaccgcc gtgtactact gcgccagggc cggcttccac 300

cccgacaact ggggccaggg caccctggtg accgtgagca gc 342

<210> 23

<211> 107

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 69 light chain variable districts

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asp Ser Asn Arg Pro Ser Gly Tyr Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

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

85 90 95

Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 24

<211> 321

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 69 light chain variable districts

<400> 24

gacatcgagc tgacccagcc ccccagcgtg agcgtggccc ccggccagac cgccaggatc 60

agctgcagcg gcgacagcat ccccaactac agcgtgagct ggtaccagca gaagcccggc 120

caggcccccg tgctggtgat ctacgccgac agcaacaggc ccagcggcta ccccgagagg 180

ttcagcggca gcaacagcgg caacaccgcc accctgacca tcagcggcac ccaggccgag 240

gacgaggccg actactactg cgagagctac gacaacacca gccccgacct gttcggcggc 300

ggcaccaagc tgaccgtgct g 321

<210> 25

<211> 116

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 81 weight chain variable districts

<400> 25

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

1 5 10 15

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

20 25 30

Tyr Ser Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Arg Asp Tyr Arg Phe Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 26

<211> 348

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 81 weight chain variable districts

<400> 26

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagc 348

<210> 27

<211> 108

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 81 light chain variable districts

<400> 27

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

1 5 10 15

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

20 25 30

Lys Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Glu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 28

<211> 324

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 81 weight chain variable districts

<400> 28

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc 60

ctgagctgca gggccagcaa gggcgtgagc gacggcaaga cctacctgta ctggtaccag 120

cagaagcccg gccaggcccc caggctgctg atctacgagg ccagctacct ggagagcggc 180

gtgcccgcca ggttcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cacagcaggg acctgcccta caccttcggc 300

ggcggcacca aggtggagat caag 324

<210> 29

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 76 weight chain variable districts

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Phe His Pro

85 90 95

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

100 105

<210> 30

<211> 807

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 76 weight chain variable districts

<400> 30

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagcgc cagcaccaag 360

ggccccagcg tgttccccct ggccccctgc agcaggagca ccagcgagag caccgccgcc 420

ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc 480

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcggcag cctgaggctg 540

agctgcgccg ccggcttcag cttcaacgcc tacgccatga gctgggtgag gcaggccccc 600

ggccagggcc tggagtgggt gagcctgatc agcaccggca gcgccaccta ctacgccgac 660

agcctgaagg gcaggttcac catcagcagg gacaacagca agaacaccct gtacctgcag 720

ttcgacgaca ccgccgtgta ctactgcgcc agggccggct tccaccccga caactggggc 780

cagggcaccc tggtgaccgt gagcagc 807

<210> 31

<211> 109

<212> PRT

<213>Artificial sequence

<220>

<223>Clone the amino acid sequence of 76 light chain variable districts

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asp Ser Asn Arg Pro Ser Gly Tyr Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

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

85 90 95

Leu Phe Gly Gly Gly Thr Lys Val Glu Gly Phe Arg Val

100 105

<210> 32

<211> 327

<212> DNA

<213>Artificial sequence

<220>

<223>Clone the nucleotide sequence of 76 light chain variable districts

<400> 32

gacatcgagc tgacccagcc ccccaccctg agcctggccc ccggccagac cgccaggatc 60

agctgcagcg gcgacagcat ccccaactac agcgtgagct ggtaccagca gaagcccggc 120

caggccccca ggctgctgat ctacgccgac agcaacaggc ccagcggcta ccccgagagg 180

ttcagcggca gcaacagcgg caccgacttc accctgacca tcagcggcac cgagcccgag 240

gacgaggccg actactactg cgagagctac gacaacacca gccccgacct gttcggcggc 300

ggcaccaagg tggagggctt cagggtg 327

<210> 33

<211> 443

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of anti-PD-1 specific antibodies heavy chain

<400> 33

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

1 5 10 15

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

20 25 30

Tyr Ser Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Arg Asp Tyr Arg Phe Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val

100 105 110

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

115 120 125

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 34

<211> 1335

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide coding sequence of the heavy chain of anti-PD-1 specific antibodies

<400> 34

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagcgc cagcaccaag 360

ggccccagcg tgttccccct ggccccctgc agcaggagca ccagcgagag caccgccgcc 420

ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc 480

gccctgacca gcggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc 540

ctgagcagcg tggtgaccgt gcccagcagc agcctgggca ccaagaccta cacctgcaac 600

gtggaccaca agcccagcaa caccaaggtg gacaagaggg tggagagcaa gtacggcccc 660

ccctgccccc cctgccccgc ccccgagttc ctgggcggcc ccagcgtgtt cctgttcccc 720

cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg 780

gacgtgagcc aggaggaccc cgaggtgcag ttcaactggt acgtggacgg cgtggaggtg 840

cacaacgcca agaccaagcc cagggaggag cagttcaaca gcacctacag ggtggtgagc 900

gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgagc 960

aacaagggcc tgcccagcag catcgagaag accatcagca aggccaaggg ccagcccagg 1020

gagccccagg tgtacaccct gccccccagc caggaggaga tgaccaagaa ccaggtgagc 1080

ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 1140

ggccagcccg agaacaacta caagaccacc ccccccgtgc tggacagcga cggcagcttc 1200

ttcctgtaca gcaggctgac cgtggacaag agcaggtggc aggagggcaa cgtgttcagc 1260

tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgagc 1320

ctgggcaagt gataa 1335

<210> 35

<211> 214

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of anti-PD-1 specific antibodies light chain

<400> 35

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

1 5 10 15

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

20 25 30

Lys Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Glu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 36

<211> 648

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide sequence of anti-PD-1 specific antibodies light chain

<400> 36

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc 60

ctgagctgca gggccagcaa gggcgtgagc gacggcaaga cctacctgta ctggtaccag 120

cagaagcccg gccaggcccc caggctgctg atctacgagg ccagctacct ggagagcggc 180

gtgcccgcca ggttcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cacagcaggg acctgcccta caccttcggc 300

ggcggcacca aggtggagat caagaccgtg gccgccccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gctaataa 648

<210> 37

<211> 468

<212> PRT

<213>Artificial sequence

<220>

<223>Anti- PD-1 scFv-Fc fusion protein amino acid sequence

<400> 37

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

1 5 10 15

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

20 25 30

Lys Thr Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Leu Leu Ile Tyr Glu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

115 120 125

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

130 135 140

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

145 150 155 160

Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Asn Pro

165 170 175

Ser Asn Gly Thr Ala Tyr Asn Pro Ala Leu Lys Arg Val Thr Leu Thr

180 185 190

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

195 200 205

Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Asp Tyr Arg Phe

210 215 220

Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu

225 230 235 240

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

305 310 315 320

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

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Leu Gly Lys

465

<210> 38

<211> 1410

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide sequence of anti-PD-1 scFv-Fc fusion proteins

<400> 38

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc 60

ctgagctgca gggccagcaa gggcgtgagc gacggcaaga cctacctgta ctggtaccag 120

cagaagcccg gccaggcccc caggctgctg atctacgagg ccagctacct ggagagcggc 180

gtgcccgcca ggttcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cacagcaggg acctgcccta caccttcggc 300

ggcggcacca aggtggagat caagggcggc ggcggcagcg gcggcggcgg cagcggcggc 360

ggcggcagcc aggtgcagct ggtgcagagc ggcgtggagg tgaagaagcc cggcgccagc 420

gtgaaggtga gctgcaaggc cagcggctac accttcacca gcgactacag ctactgggtg 480

aggcaggccc ccggccaggg cctggagtgg atgggcggca tcaaccccag caacggcacc 540

gcctacaacc ccgccctgaa gagggtgacc ctgaccaccg acagcagcac caccaccgcc 600

tacatggagc tgaagagcct gcagttcgac gacaccgccg tgtactactg cgccaggagg 660

gactacaggt tcgacatggg cgacctgggc cagggcacca ccgtgaccgt gagcagcgag 720

agcaagtacg gccccccctg ccccccctgc cccgcccccg agttcctggg cggccccagc 780

gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg 840

acctgcgtgg tggtggacgt gagccaggag gaccccgagg tgcagttcaa ctggtacgtg 900

gacggcgtgg aggtgcacaa cgccaagacc aagcccaggg aggagcagtt caacagcacc 960

tacagggtgg tgagcgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 1020

aagtgcaagg tgagcaacaa gggcctgccc agcagcatcg agaagaccat cagcaaggcc 1080

aagggccagc ccagggagcc ccaggtgtac accctgcccc ccagccagga ggagatgacc 1140

aagaaccagg tgagcctgac ctgcctggtg aagggcttct accccagcga catcgccgtg 1200

gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac 1260

agcgacggca gcttcttcct gtacagcagg ctgaccgtgg acaagagcag gtggcaggag 1320

ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380

agcctgagcc tgagcctggg caagtgataa 1410

<210> 39

<211> 436

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of anti-TEM8 heavy chains

<400> 39

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Phe His Pro

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

180 185 190

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

195 200 205

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

275 280 285

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

290 295 300

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Ser Leu Gly Lys

435

<210> 40

<211> 1356

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide sequence of anti-TEM8 heavy chains

<400> 40

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcggcag cctgaggctg 60

agctgcgccg ccggcttcag cttcaacgcc tacgccatga gctgggtgag gcaggccccc 120

ggccagggcc tggagtgggt gagcctgatc agcaccggca gcgccaccta ctacgccgac 180

agcctgaagg gcaggttcac catcagcagg gacaacagca agaacaccct gtacctgcag 240

ttcgacgaca ccgccgtgta ctactgcgcc agggccggct tccaccccga caactggggc 300

cagggcaccc tggtgaccgt gagcagcgcc agcaccaagg gccccagcgt gttccccctg 360

gccccctgca gcaggagcac cagcgagagc accgccgccc tgggctgcct ggtgaaggac 420

tacttccccg agcccgtgac cgtgagctgg aacagcggcg ccctgaccag cggcgtgcac 480

accttccccg ccgtgctgca gagcagcggc ctgtacagcc tgagcagcgt ggtgaccgtg 540

cccagcagca gcctgggcac caagacctac acctgcaacg tggaccacaa gcccagcaac 600

accaaggtgg acaagagggt ggagagcaag tacggccccc cctgcccccc ctgccccgcc 660

cccgagttcc tgggcggccc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 720

atgatcagca ggacccccga ggtgacctgc gtggtggtgg acgtgagcca ggaggacccc 780

gaggtgcagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 840

agggaggagc agttcaacag cacctacagg gtggtgagcg tgctgaccgt gctgcaccag 900

gactggctga acggcaagga gtacaagtgc aaggtgagca acaagggcct gcccagcagc 960

atcgagaaga ccatcagcaa ggccaagggc cagcccaggg agccccaggt gtacaccctg 1020

ccccccagcc aggaggagat gaccaagaac caggtgagcc tgacctgcct ggtgaagggc 1080

ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac 1140

aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctgtacag caggctgacc 1200

gtggacaaga gcaggtggca ggagggcaac gtgttcagct gcagcgtgat gcacgaggcc 1260

ctgcacaacc actacaccca gaagagcctg agcctgagcc tgggcaagtg ataaagcctg 1320

agcctgagcc tgggcaagtg ataaggcaag tgataa 1356

<210> 41

<211> 277

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of anti-TEM8 light chains

<400> 41

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asp Ser Asn Arg Pro Ser Gly Tyr Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

115 120 125

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

130 135 140

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

145 150 155 160

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

165 170 175

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

180 185 190

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

195 200 205

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

210 215 220

Phe Ala Val Tyr Tyr Cys Gln His Ser Arg Asp Leu Pro Tyr Thr Phe

225 230 235 240

Gly Gly Gly Thr Lys Val Glu Ile Lys Tyr Phe Cys Phe Gln Gly Ile

245 250 255

His Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr

260 265 270

Val Ser Ser Leu Lys

275

<210> 42

<211> 765

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide sequence of anti-TEM8 light chains

<400> 42

gacatcgagc tgacccagcc ccccaccctg agcctggccc ccggccagac cgccaggatc 60

agctgcagcg gcgacagcat ccccaactac agcgtgagct ggtaccagca gaagcccggc 120

caggccccca ggctgctgat ctacgccgac agcaacaggc ccagcggcta ccccgagagg 180

ttcagcggca gcaacagcgg caccgacttc accctgacca tcagcggcac cgagcccgag 240

gacgaggccg actactactg cgagagctac gacaacacca gccccgacct gttcggcggc 300

ggcaccaagg tggagggctt cagggtgagc gacgagcagc tgaagagcgg caccgccagc 360

gtggtgtgcc tgctgaacaa cttctacccc agggaggcca aggtgcagtg gaaggtggac 420

aacgccctgc agagcggcaa cagccaggag agcgtgaccg agcaggacag caaggacagc 480

acctacagcc tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg 540

tacgcctgcg aggtgaccca ccagggcctg agcagccccg tgaccaagag cttcaacagg 600

ggcgagtgcg ccaggttcag cggcagcggc accgacttca ccctgaccat cagcagcctg 660

gagcccgagg acttcgccgt gtactactgc cagcacagca gggacctgcc ctacaccttc 720

ggcggcggca ccaaggtgga gatcaagtga taaatcaagt gataa 765

<210> 43

<211> 462

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of anti-TEM8 scFv-Fc fusion proteins

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Asp Ser Asn Arg Pro Ser Gly Tyr Pro Glu Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

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

85 90 95

Leu Phe Gly Gly Gly Thr Lys Val Glu Gly Phe Arg Val Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

115 120 125

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

130 135 140

Ser Cys Ala Ala Gly Phe Ser Phe Asn Ala Tyr Ala Met Ser Trp Val

145 150 155 160

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

165 170 175

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

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Phe Asp Asp Thr

195 200 205

Ala Val Tyr Tyr Cys Ala Arg Ala Gly Phe His Pro Asp Asn Trp Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

<210> 44

<211> 1392

<212> DNA

<213>Artificial sequence

<220>

<223>The nucleotide sequence of anti-TEM8 scFv-Fc fusion proteins

<400> 44

gacatcgagc tgacccagcc ccccaccctg agcctggccc ccggccagac cgccaggatc 60

agctgcagcg gcgacagcat ccccaactac agcgtgagct ggtaccagca gaagcccggc 120

caggccccca ggctgctgat ctacgccgac agcaacaggc ccagcggcta ccccgagagg 180

ttcagcggca gcaacagcgg caccgacttc accctgacca tcagcggcac cgagcccgag 240

gacgaggccg actactactg cgagagctac gacaacacca gccccgacct gttcggcggc 300

ggcaccaagg tggagggctt cagggtgggc ggcggcggca gcggcggcgg cggcagcggc 360

ggcggcggca gccaggtgca gctggtgcag agcggcgtgg aggtgaagaa gcccggcggc 420

agcctgaggc tgagctgcgc cgccggcttc agcttcaacg cctacgccat gagctgggtg 480

aggcaggccc ccggccaggg cctggagtgg gtgagcctga tcagcaccgg cagcgccacc 540

tactacgccg acagcctgaa gggcaggttc accatcagca gggacaacag caagaacacc 600

ctgtacctgc agttcgacga caccgccgtg tactactgcg ccagggccgg cttccacccc 660

gacaactggg gccagggcac cctggtgacc gtgagcagcg agagcaagta cggccccccc 720

tgccccccct gccccgcccc cgagttcctg ggcggcccca gcgtgttcct gttccccccc 780

aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggac 840

gtgagccagg aggaccccga ggtgcagttc aactggtacg tggacggcgt ggaggtgcac 900

aacgccaaga ccaagcccag ggaggagcag ttcaacagca cctacagggt ggtgagcgtg 960

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtgagcaac 1020

aagggcctgc ccagcagcat cgagaagacc atcagcaagg ccaagggcca gcccagggag 1080

ccccaggtgt acaccctgcc ccccagccag gaggagatga ccaagaacca ggtgagcctg 1140

acctgcctgg tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc 1200

cagcccgaga acaactacaa gaccaccccc cccgtgctgg acagcgacgg cagcttcttc 1260

ctgtacagca ggctgaccgt ggacaagagc aggtggcagg agggcaacgt gttcagctgc 1320

agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgag cctgagcctg 1380

ggcaagtgat aa 1392

<210> 45

<211> 686

<212> PRT

<213>Artificial sequence

<220>

<223>Anti- PD-1 heavy chains-TEM8scFv amino acid sequence

<400> 45

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

1 5 10 15

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

20 25 30

Tyr Ser Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Arg Asp Tyr Arg Phe Asp Met Gly Asp Leu Gly Gln Gly Thr Thr Val

100 105 110

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

115 120 125

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser

435 440 445

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

450 455 460

Leu Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Ile

465 470 475 480

Pro Asn Tyr Ser Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

485 490 495

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

500 505 510

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

515 520 525

Gly Thr Glu Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Glu Ser Tyr Asp

530 535 540

Asn Thr Ser Pro Asp Leu Phe Gly Gly Gly Thr Lys Val Glu Gly Phe

545 550 555 560

Arg Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Phe His

660 665 670

Pro Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

675 680 685

<210> 46

<211> 2064

<212> DNA

<213>Artificial sequence

<220>

<223>Anti- PD-1 heavy chains-TEM8scFv nucleotide sequence

<400> 46

caggtgcagc tggtgcagag cggcgtggag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agcgactaca gctactgggt gaggcaggcc 120

cccggccagg gcctggagtg gatgggcggc atcaacccca gcaacggcac cgcctacaac 180

cccgccctga agagggtgac cctgaccacc gacagcagca ccaccaccgc ctacatggag 240

ctgaagagcc tgcagttcga cgacaccgcc gtgtactact gcgccaggag ggactacagg 300

ttcgacatgg gcgacctggg ccagggcacc accgtgaccg tgagcagcgc cagcaccaag 360

ggccccagcg tgttccccct ggccccctgc agcaggagca ccagcgagag caccgccgcc 420

ctgggctgcc tggtgaagga ctacttcccc gagcccgtga ccgtgagctg gaacagcggc 480

gccctgacca gcggcgtgca caccttcccc gccgtgctgc agagcagcgg cctgtacagc 540

ctgagcagcg tggtgaccgt gcccagcagc agcctgggca ccaagaccta cacctgcaac 600

gtggaccaca agcccagcaa caccaaggtg gacaagaggg tggagagcaa gtacggcccc 660

ccctgccccc cctgccccgc ccccgagttc ctgggcggcc ccagcgtgtt cctgttcccc 720

cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg 780

gacgtgagcc aggaggaccc cgaggtgcag ttcaactggt acgtggacgg cgtggaggtg 840

cacaacgcca agaccaagcc cagggaggag cagttcaaca gcacctacag ggtggtgagc 900

gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgagc 960

aacaagggcc tgcccagcag catcgagaag accatcagca aggccaaggg ccagcccagg 1020

gagccccagg tgtacaccct gccccccagc caggaggaga tgaccaagaa ccaggtgagc 1080

ctgacctgcc tggtgaaggg cttctacccc agcgacatcg ccgtggagtg ggagagcaac 1140

ggccagcccg agaacaacta caagaccacc ccccccgtgc tggacagcga cggcagcttc 1200

ttcctgtaca gcaggctgac cgtggacaag agcaggtggc aggagggcaa cgtgttcagc 1260

tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagagcct gagcctgagc 1320

ctgggcaagg gcggcggcgg cagcggcggc ggcggcagcg acatcgagct gacccagccc 1380

cccaccctga gcctggcccc cggccagacc gccaggatca gctgcagcgg cgacagcatc 1440

cccaactaca gcgtgagctg gtaccagcag aagcccggcc aggcccccag gctgctgatc 1500

tacgccgaca gcaacaggcc cagcggctac cccgagaggt tcagcggcag caacagcggc 1560

accgacttca ccctgaccat cagcggcacc gagcccgagg acgaggccga ctactactgc 1620

gagagctacg acaacaccag ccccgacctg ttcggcggcg gcaccaaggt ggagggcttc 1680

agggtgggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag ccaggtgcag 1740

ctggtgcaga gcggcgtgga ggtgaagaag cccggcggca gcctgaggct gagctgcgcc 1800

gccggcttca gcttcaacgc ctacgccatg agctgggtga ggcaggcccc cggccagggc 1860

ctggagtggg tgagcctgat cagcaccggc agcgccacct actacgccga cagcctgaag 1920

ggcaggttca ccatcagcag ggacaacagc aagaacaccc tgtacctgca gttcgacgac 1980

accgccgtgt actactgcgc cagggccggc ttccaccccg acaactgggg ccagggcacc 2040

ctggtgaccg tgagcagctg ataa 2064

Claims (11)

1. a kind of anti-PD-1 and TEM-8 bispecific antibodies or its variant or its functional fragment, it includes：

A. specific recognition and immune cell surface antigenic PD-1 domain is combined, it includes the weight of anti-PD-1 specific antibodies Chain variable region (anti-PD-1VH)；And

B. specific recognition and tumor endothelial mark TEM-8 domain is combined, it includes the weight of anti-TEM-8 specific antibodies Chain variable region (anti-TEM-8VH).

2. anti-PD-1 and TEM-8 bispecific antibodies according to claim 1 or its variant or its functional fragment, institute State specific recognition and combine immune cell surface antigenic PD-1 domain by the anti-PD-1 specific antibodies that are sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and Fc fragments (anti-PD-1CH2-CH3) composition, Or by light chain (anti-PD-1VL-CL) and heavy chain (the anti-PD-1VH-CH1- hinge areas-CH2- of one group of anti-PD-1 specific antibody CH3) constitute, or by the light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinges of two groups of anti-PD-1 specific antibodies Area-CH2-CH3) composition, or by the light chain variable district (anti-PD-1VL) and weight chain variable district (anti-PD- of anti-PD-1 specific antibodies 1VH) constitute；

Preferably, the specific recognition and tumor endothelial mark TEM-8 domain is combined by the anti-TEM-8 that is sequentially connected Light chain variable district (anti-TEM-8VL), weight chain variable district (anti-TEM-8VH), hinge area and Fc fragments (the anti-TEM- of specific antibody 8CH2-CH3) constitute, or by the light chain (anti-TEM-8VL-CL) and heavy chain (anti-TEM-8VH- of one group of anti-TEM-8 specific antibody CH1- hinge areas-CH2-CH3) composition, or by the light chain (anti-TEM-8VL-CL) and heavy chain of two groups of anti-TEM-8 specific antibodies (anti-TEM-8VH-CH1- hinge areas-CH2-CH3) is constituted, or by light chain variable district (the anti-TEM- of anti-TEM-8 specific antibodies 8VL) constituted with weight chain variable district (anti-TEM-8VH).

3. anti-PD-1 and TEM-8 bispecific antibodies according to claim 1 or 2 or its variant or its feature piece Section,

It includes：

A ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specific antibody Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition；And

B ' specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is by one group of anti-TEM-8 specific antibody Light chain (anti-TEM-8VL-CL) and heavy chain (anti-TEM-8VH-CH1- hinge areas-CH2-CH3) composition；

Preferably, the specific recognition and domain with reference to immune cell surface antigenic PD-1 and the specific recognition and With reference to tumor endothelial mark TEM-8 domain by one or more disulfide bond, such as it is one or more to be located at hinge area Disulfide bond；Or

It includes：

A " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by one group of anti-PD-1 specific antibody Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition；And

B " specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is special by the anti-TEM-8 being sequentially connected Light chain variable district (anti-TEM-8VL), weight chain variable district (anti-TEM-8VH), hinge area and Fc fragments (the anti-TEM- of property antibody 8CH2-CH3) constitute；

Preferably, the specific recognition and domain with reference to immune cell surface antigenic PD-1 and the specific recognition and With reference to tumor endothelial mark TEM-8 domain by one or more disulfide bond, such as it is one or more to be located at hinge area Disulfide bond；Or

It includes：

A " ' specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is special by the anti-PD-1 being sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and Fc fragments (the anti-PD-1CH2- of property antibody CH3) constitute；And

B " ' specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is by one group of anti-TEM-8 specific antibody Light chain (anti-TEM-8VL-CL) and heavy chain (anti-TEM-8VH-CH1- hinge areas-CH2-CH3) composition；

Preferably, the specific recognition and domain with reference to immune cell surface antigenic PD-1 and the specific recognition and With reference to tumor endothelial mark TEM-8 domain by one or more disulfide bond, such as it is one or more to be located at hinge area Disulfide bond；Or

It includes：

A " " specific recognitions and the domain for combining immune cell surface antigenic PD-1, it is by two groups of anti-PD-1 specific antibodies Light chain (anti-PD-1VL-CL) and heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) composition；And

B " " specific recognitions and the domain for combining tumor endothelial mark TEM-8, it is by two groups of anti-TEM-8 specific antibodies Light chain variable district (anti-TEM-8VL) and weight chain variable district (anti-TEM-8VH) composition；

Preferably, each anti-PD-1 in the specific recognition and combination immune cell surface antigenic PD-1 domain is special The CH3 of the heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) of property antibody is respectively with the specific recognition and combining tumour The weight chain variable district (anti-TEM-8VH) of each anti-TEM-8 specific antibodies in endothelial marker TEM-8 domain is by changing Learn key connection.

4. anti-PD-1 and TEM-8 bispecific antibodies according to any one of claim 1 to 3 or its variant or its work( Energy property fragment, it is chimeric antibody, humanized antibody or human antibody.

5. anti-PD-1 and TEM-8 bispecific antibodies according to any one of claim 1 to 4 or its variant or its work( Energy property fragment, wherein the amino acid sequence such as SEQ of the weight chain variable district in the specific recognition and combination PD-1 domain ID NO:1st, shown in 5,9 or 25；The amino acid sequence of light chain variable district such as SEQ ID NO:3rd, shown in 7,11 or 27；

Preferably, the amino acid sequence such as SEQ of the weight chain variable district in the specific recognition and combination TEM-8 domain ID NO:13rd, shown in 17,21 or 29；The amino acid sequence of light chain variable district such as SEQ ID NO:15th, shown in 19,23 or 31.

Preferably, the amino acid sequence of the heavy chain (anti-PD-1VH-CH1- hinge areas-CH2-CH3) of the anti-PD-1 specific antibodies It is classified as SEQ ID No:33, nucleotides sequence is classified as SEQ ID No:34；Light chain (the anti-PD- of the anti-PD-1 specific antibodies Amino acid sequence 1VL-CL) is SEQ ID No:35, nucleotides sequence is classified as SEQ ID No:36；It is described anti-by what is be sequentially connected Light chain variable district (anti-PD-1VL), weight chain variable district (anti-PD-1VH), hinge area and the Fc fragments of PD-1 specific antibodies (resist PD-1CH2-CH3) the amino acid sequence of the specific recognition of composition and combination immune cell surface antigenic PD-1 domain It is classified as SEQ ID No:37, nucleotides sequence is classified as SEQ ID No:38；

Preferably, the amino acid of the heavy chain (anti-TEM-8VH-CH1- hinge areas-CH2-CH3) of the anti-TEM-8 specific antibodies Sequence is SEQ ID No:39, nucleotides sequence is classified as SEQ ID No:40；The light chain of the anti-TEM-8 specific antibodies is (anti- TEM-8VL-CL amino acid sequence) is SEQ ID No:41, nucleotides sequence is classified as SEQ ID No:42；It is described by being sequentially connected Anti- TEM-8 specific antibodies light chain variable district (anti-TEM-8VL), weight chain variable district (anti-TEM-8VH), hinge area and Fc pieces The amino of the specific recognition of section (anti-TEM-8CH2-CH3) composition and combination tumor endothelial mark TEM-8 domain Acid sequence is SEQ ID No:43, nucleotides sequence is classified as SEQ ID No:44；The light chain by anti-TEM-8 specific antibodies can Become area (anti-TEM-8VL) and weight chain variable district (anti-TEM-8VH) the composition specific recognition and combine tumor endothelial mark The amino acid sequence of thing TEM-8 domain is SEQ ID No:45, nucleotides sequence is classified as SEQ ID No:46；

Preferably, the nucleotide sequence such as SEQ ID of the weight chain variable district in the specific recognition and combination PD-1 domain NO:2nd, shown in 6,10 or 26；The nucleotide sequence of light chain variable district such as SEQ ID NO:4th, shown in 8,12 or 28；

Preferably, the nucleotide sequence such as SEQ of the weight chain variable district in the specific recognition and combination TEM-8 domain ID NO:14th, shown in 18,22 or 30；The nucleotide sequence of light chain variable district such as SEQ ID NO:16th, shown in 20,24 or 32.

6. one kind coding anti-PD-1 and TEM-8 bispecific antibodies according to any one of claim 1 to 5 or its change The nucleic acid molecules of body or its functional fragment.

7. a kind of expression vector for including nucleic acid molecules according to claim 6.

8. a kind of host cell for including expression vector according to claim 7.

9. a kind of pharmaceutical composition, it is double special that it includes anti-PD-1 and TEM-8 according to any one of claim 1 to 5 Property antibody or its variant or its functional fragment, either nucleic acid molecules according to claim 6 or will according to right Ask the expression vector described in 7, or host cell according to claim 8；

Preferably, described pharmaceutical composition also includes pharmaceutically useful carrier and/or auxiliary material；Preferably, described pharmaceutical composition is also Including other activating agents, such as chemotherapeutics, such as Tegafur, fluorouracil and oxaliplatin；Cancer adjuvant therapy medicament, such as Thymosin alpha 1, thymopeptide-5, Calciumlevofolinate, Granisetron and Tropisetron etc.；Cytotoxic agent, such as Ali's statin, calicheamicin, Maytansine and times carcinomycin etc.；Immunocyte, such as NK cells, T cell and DC-CIK cells etc.；

Preferably, the formulation of described pharmaceutical composition can be liquid injection formulation or solid suppository formulation.

10. anti-PD-1 and TEM-8 bispecific antibodies according to any one of claim 1 to 5 or its variant or its Functional fragment, nucleic acid molecules according to claim 6, expression vector according to claim 7 or according to right It is required that host cell described in 8 and making immune increasing by suppressing the immunosupress such as PD-1, PD-L1 or PD-L2 signal path Applications of the strong joint targeting TEM-8 in the medicine for the treatment of cancer, infectious diseases or inflammatory disease；

Specifically, wherein the cancer is selected from stomach cancer, carcinoma of testis, uterine cancer, carcinoma of fallopian tube, carcinoma of endometrium, cervical carcinoma, vagina Cancer, cancer of the esophagus, carcinoma of small intestine, thyroid cancer, parathyroid carcinoma, melanoma, kidney, prostate cancer, breast cancer, colon cancer, lung cancer, bone Cancer, cancer of pancreas, cutaneum carcinoma, incidence cancer, skin or intraocular chromoma, uterine cancer, oophoroma, the carcinoma of the rectum, adrenal, Cancer of the anal region, vaginal orifice cancer, carcinoma of urethra, carcinoma of penis, carcinoma of urinary bladder, kidney or carcinoma of ureter, carcinoma of renal pelvis, epidermoid carcinoma, squamous cell carcinoma, what Outstanding king's evil, non_hodgkin lymphoma, the cancer of internal system, soft tissue sarcoma, the neoplasm of central nervous system, original Send out sexual centre nervous system lymthoma, tumor vessel generation, spinal cord axle tumour, brain stem glioma, pituitary adenoma, Ka Boxishi meat Knurl, t cell lymphoma, chronic or acute leukemia (it include acute myeloid leukemia, chronic myeloid leukemia, Acute lymphatic leukemia, chronic lymphocytic leukemia), childhood solid tumor, in lymphocytic lymphoma It is one or more；

The infectious diseases is selected from HIV, and influenza, bleb, Giardiasis, malaria, leishmaniasis, or following virus are drawn The infection risen：Hepatitis viruse (such as A type, B-mode or HCV), herpesviral (such as VZV, HSV-1, HAV-6, HSV-II, CMV or angstrom bar Er Shi virus), adenovirus, influenza virus, vaccinia virus, HTLV viruses, dengue fever virus, papilloma Virus, contagiosum, poliovirus, hydrophobin, flavivirus, echovirus, rhinovirus, Coxsackie virus, hat Shape virus, Respiratory Syncytial Virus(RSV), mumps virus, rotavirus measles virus, rubella virus, parvovirus, JC virus and Arboviral encephalitides virus, or following bacterial infection：Chlamydia, rickettsia, pneumococcus, mycobacteria, Staphylococcus, streptococcus, meningococcus, conococci, Serratieae, Klebsiella, mycetozoan, pseudomonad, sramana Salmonella, comma bacillus, corynebacterium diphtheriae, clostridium botulinum, bacillus anthracis, clostridium tetani, Legionella, yersinia pestis, hook end spiral Body disease or Lyme disease bacterium, below fungus-caused infection：Aspergillus (such as aspergillus fumigatus, aspergillus niger), Candida are (for example Candida albicans), candida krusei, Candida glabrata, candida tropicalis, Cryptococcus neoformans, dermatitis bud ferment Female, category (such as mucor, Absidia or rhizopus), sporotrichum schenckii, Paracoccidioides brasiliensis, the thick ball of Mucoales Spore bacterium or blooming histoplasma capsulatum, infection caused by following parasite：Entamoeba historlytica, colon bowel bag worm, good fortune Na Shi worms, spine Amoeba, Plasmodium vivax, Babesiamicrofti, suck giardia lamblia, Cryptosporidium, Pneumocystis carinii, T. brucei, Cruz Trypanosome, Duo Shi Leishmanias, mouse Earth's bow shock or nippostrongylus brasiliensis；

The inflammatory disease is selected from acute diseminated encephalomyelitis, Addision's disease, ankylosing spondylitis, anti-phospholipid antibody and integrated Levy, autoimmune hemolytic anemia, oneself immunity hepatitis, arthritis, behcet's disease, american trypanosomiasis, Crohn disease, bleb Property pemphigoid, abdominal disease, dermatomyositis, graft versus host disease(GVH disease), Graves disease, type 1 diabetes, empsyxis-ephritis are comprehensive It is simulator sickness, guillain-Barre syndrome, chronic lymphocytic thyroiditis, super IgE syndromes, ITP, lupus erythematosus, multiple Property sclerosis, myasthenia gravis, pemphigus, pernicious anaemia, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid close Save inflammation, xerodermosteosis, temporal arteritis, vasculitis or Wegner's granulomatosis.

11. a kind of anti-PD-1 and TEM-8 bispecific antibodies or its variant according to any one of claim 1 to 5, Or the preparation method of its functional fragment, it, which is included in, can produce the anti-PD-1 and TEM-8 bispecific antibodies or its change Host cell according to claim 8 is cultivated under conditions of body or its functional fragment, and reclaims produced resist PD-1 and TEM-8 bispecific antibodies or its variant or its functional fragment.