CN106986939B - 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 antibody and its application

Technical field

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

Background technique

Bispecific antibody refers to the artificial antibody containing 2 species-specific antigen binding sites, can in target cell and Bridge is erected between functional molecular (cell), is excited the immune response with guiding performance, is one kind of genetic engineering antibody.Double spies Heterogenetic antibody has become the hot spot in antibody engineering field, has broad application prospects in the immunization therapy of tumour.Currently, There are mainly three types of methods for the preparation of this antibody: chemical coupling method, slush pump underlayer method and genetic engineering antibody the preparation method, Middle genetic engineering antibody the preparation method is the most frequently used, also most advanced.In general, it is big to be segmented into two for bispecific binding protein molecule Class: one kind is containing the area Fc, and another kind of to be a lack of the area Fc, the latter is typically less than immunoglobulin and IgG containing the area Fc is bis- Specific molecular.The area Fc is conducive to purify bispecific antibody using the established process for IgG molecule, and And its dissolubility and stability can be improved.In addition, the area Fc also has an impact to the Fc- effector function mediated, this may be some controls It treats and applies required additive effect, such as the cytotoxicity (ADCC) of antibody-dependant, complement combine (CDC) and since it has more Long half-lift caused by the cyclic process that big molecular weight and Fc neonatal receptor (FcRn) mediate.These functions can pass through The means of genetic engineering are further utilized, for example, retaining long half-lift simultaneously by eliminating ADCC and/or CDC.Compared to it Under, the bispecific binding protein molecule for lacking the area Fc is completely dependent on its antigen binding capacity to play treatment effect.

Programmed cell death factors 1 (programmed death 1, PD-1) are one kind mainly in activating immune cell The Inhibitory receptor of upper expression.In tumor patient, negativity adjustment signal, ginseng are immunized by the high expression mediate T cell of PD-1 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 ligand 2 (programmed death 2-ligand, PD-L2) be PD-1 binding partner (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 in conjunction with PD-1 extracellular region, die body is inhibited by the immunity receptor tyrosine of PD-1 cytoplasmic region tail portion, The phosphatidylinositol3 3 kinase of CD3 (CD28) induction and the activity of protein kinase B are lowered, turning for T cell proliferation-associated genes is inhibited It records and promotes t cell proliferation；Protein-tyrosine-phosphatase 1 is also recruited simultaneously, makes signaling molecule dephosphorylation in connection, from And inhibit 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 relevant 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), ligand 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, gastric cancer, clear-cell carcinoma), shows PD-1/PD- L signal access 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 It can be by inhibiting that anticancer therapy is effectively performed and immune response in tumor environment.Over the past two years, U.S. clinical tumour annual meeting Exciting clinical test is probably exactly PD-1 anticancrin bring black whirlwind.PD-1 antibody bring clinical anti-cancer Effect is unprecedented.It can control the cancer progression of 50% cutaneum carcinoma patient, cure 10% or so skin carninomatosis People.It is medically helpless for many years for obstinate non-small cell lung cancer patient, but PD-1 antibody has and faces to 24% patient Bed control effect.Since its broad spectrum anticancer acts on, to kidney, gastric cancer, breast cancer, bladder cancer, leukemia, head and neck cancer, intestinal cancer and brain The clinical efficacy of the cancers such as tumor is also among test of clinical the second stage of or three phases.

Tumor endothelial marker (tumor endothelia marker, TEM 1-9) is by tumor blood vessels Chrotoplast and the gene expression for closing on Normal tissue vascular endothelial cell carry out gene series winding and compare analysis (serial Analysis gene express, SAGE) discovery 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 family, compared with other TEM and VEGFR, TEM- 8 is more special in tumor vascular endothelium expression, exists in tumor vessel, and seldom or is not present in normal tissue.More attach most importance to It wants, TEM-8 is then accredited as a kind of receptor of anthrax toxin.TEM-8 itself has important biological function, high The TEM-8 of expression can promote the adherency and migration of cell, but the extracellular region protein that these effects can be recombinated blocks (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 Expression (Rmali KA, et al., the Upregulation of tumor endothelial marker-8 of aobvious induction TEM-8 by interleukin-1βand its impact in IL-1βinduced angiogenesis.Int J Mol Med, 2004,14(1):75-80).In addition, being directed to the anti-tumour antibody sample albumen of TEM-8, i.e. people TEM-8 extracellular region and human IgG FC The fusion protein TEM-8 FC of segment has inhibits tumour growth activity and antitumous effect significantly, illustrates that TEM-8 is one 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 drug has powerful broad spectrum anticancer living kinds of tumors since it targets immunologic test point Property, but there is also a variety of potential side effects simultaneously, and although single target antibody therapy initially there are certain treatment Effect, would generally generate the treatment resistance caused by the up-regulation of other signals transduction cascade in subsequent treatment, it is therefore necessary to PD-1 antibody drug antitumor targeting ability is further increased, the risk for the treatment of side effect is reduced, while improving validity.And with The fast development of computer science, in recent years Computer-Aided Drug Design become research and development new drug brand-new technology, Obviously accelerate the speed and efficiency of new drug design.Pharmaceutical grade protein based on protein steric structure is studied by attention, egg The continuous parsing of white matter structure, PDB data constantly increase, and make it possible CAD pharmaceutical grade protein.

Summary of the invention

For defect existing for PD-1 antibody drug in the prior art, technical problem to be solved by the invention is to provide one Kind has the novel PD-1 antibody drug of the anticancer activity significantly improved.By a large amount of screening test and optimizing research, this hair Bright people have been surprisingly found that a kind of with the anti-tumor target tropism significantly improved, reduced treatment side effect risk, simultaneously With the anti-PD-1 of the validity significantly improved and the bispecific antibody of TEM-8.Most of all, the experimental results showed that, with The antibody of other tumor endothelial markers is compared, anti-PD-1 antibody and anti-TEM-8 antibody are combined be formed by it is double special Property antibody produce apparent synergy, i.e., its curative effect has aobvious on statistical significance compared to the curative effect of two kinds of monospecific antibodies It writes and improves, the prior art institute before this is the present invention is unforeseen.

The technical solution 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 antibody or its variant or its functional fragment comprising:

A. the structural domain of specific recognition and combination immune cell surface antigenic PD-1 comprising anti-PD-1 specificity is anti- The heavy chain variable region (anti-PD-1 VH) of body；And

B. the structural domain of specific recognition and combination tumor endothelial marker TEM-8 comprising anti-TEM-8 specificity is anti- The heavy chain variable region (anti-TEM-8 VH) of body.

Preferably, the structural domain of the specific recognition and combination immune cell surface antigenic PD-1 are by sequentially connected anti- Light chain variable region (anti-PD-1 VL), heavy chain variable region (anti-PD-1 VH), hinge area and the Fc segment of PD-1 specific antibody (resist PD-1CH2-CH3 it) forms, 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 area-CH2-CH3) composition, or the light chain (anti-PD-1 VL-CL) and again by two groups of anti-PD-1 specific antibodies Chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) composition, or by light chain variable region (the anti-PD-1 of anti-PD-1 specific antibody VL it) is formed with heavy chain variable region (anti-PD-1 VH).

Preferably, the structural domain of the specific recognition and combination tumor endothelial marker TEM-8 are by sequentially connected anti- Light chain variable region (anti-TEM-8 VL), heavy chain variable region (anti-TEM-8 VH), hinge area and the Fc piece of TEM-8 specific antibody 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 area-CH2-CH3) composition, or by light chain (the anti-TEM-8 of two groups of anti-TEM-8 specific antibodies VL-CL it) is formed with heavy chain (anti-TEM-8 VH-CH1- hinge area-CH2-CH3), or by the light chain of anti-TEM-8 specific antibody Variable region (anti-TEM-8 VL) and heavy chain variable region (anti-TEM-8 VH) composition.

A preferred embodiment according to the present invention, the anti-PD-1 and TEM-8 bispecific antibody or its change Body or its functional fragment include:

A ' specific recognition and the structural domain for combining immune cell surface antigenic PD-1, by one group of anti-PD-1 specificity The light chain (anti-PD-1 VL-CL) and heavy chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) of antibody form；And

B ' specific recognition and the structural domain for combining tumor endothelial marker TEM-8, by one group of anti-TEM-8 specificity The light chain (anti-TEM-8 VL-CL) and heavy chain (anti-TEM-8 VH-CH1- hinge area-CH2-CH3) of antibody form.

Preferably, the specific recognition and structural domain and the specificity knowledge in conjunction with immune cell surface antigenic PD-1 Not and the structural domain of tumor endothelial marker TEM-8 is combined to pass through one or more disulfide bond, such as one or more is located at hinge The disulfide bond of sequence connects.

A preferred embodiment according to the present invention, the anti-PD-1 and TEM-8 bispecific antibody or its change Body or its functional fragment include:

A " specific recognition and the structural domain for combining immune cell surface antigenic PD-1, by one group of anti-PD-1 specificity The light chain (anti-PD-1 VL-CL) and heavy chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) of antibody form；And

B " specific recognition and the structural domain for combining tumor endothelial marker TEM-8, by sequentially connected anti-TEM-8 Light chain variable region (anti-TEM-8 VL), heavy chain variable region (anti-TEM-8 VH), hinge area and the Fc segment of specific antibody (resist TEM-8 CH2-CH3) composition.

Preferably, the specific recognition and structural domain and the specificity knowledge in conjunction with immune cell surface antigenic PD-1 Not and the structural domain of tumor endothelial marker TEM-8 is combined to pass through one or more disulfide bond, such as one or more is located at hinge The disulfide bond of sequence connects.

A preferred embodiment according to the present invention, the anti-PD-1 and TEM-8 bispecific antibody or its change Body or its functional fragment include:

A " ' specific recognition and the structural domain for combining immune cell surface antigenic PD-1, by sequentially connected anti-PD-1 Light chain variable region (anti-PD-1 VL), heavy chain variable region (anti-PD-1 VH), hinge area and Fc segment (the anti-PD- of specific antibody 1CH2-CH3) form；And

B " ' specific recognition and the structural domain for combining tumor endothelial marker 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 area-CH2-CH3) composition.

Preferably, the specific recognition and structural domain and the specificity knowledge in conjunction with immune cell surface antigenic PD-1 Not and the structural domain of tumor endothelial marker TEM-8 is combined to pass through one or more disulfide bond, such as one or more is located at hinge The disulfide bond of sequence connects.

A preferred embodiment according to the present invention, the anti-PD-1 and TEM-8 bispecific antibody or its change Body or its functional fragment include:

A " " specific recognition and the structural 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 area-CH2-CH3) composition；And

B " " specific recognition and the structural domain for combining tumor endothelial marker TEM-8, it is special by two groups of anti-TEM-8 Property antibody light chain variable region (anti-TEM-8 VL) and heavy chain variable region (anti-TEM-8 VH) composition.

Preferably, the anti-PD-1 of each of the specific recognition and the structural domain for combining immune cell surface antigenic PD-1 The CH3 of the heavy chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) of specific antibody respectively with the specific recognition and knot Close heavy chain variable region (the anti-TEM-8 of the anti-TEM-8 specific antibody of each of the structural domain of tumor endothelial marker TEM-8 VH) pass through chemistry key connection.

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

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

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

Preferably, the ammonia of the heavy chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) of the anti-PD-1 specific antibody Base acid sequence is SEQ ID No:33, and nucleotides sequence is classified as SEQ ID No:34；The light chain of the anti-PD-1 specific antibody 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 successively connecting Light chain variable region (anti-PD-1 VL), heavy chain variable region (anti-PD-1 VH), hinge area and the Fc of the anti-PD-1 specific antibody connect The structural domain of the specific recognition and combination immune cell surface antigenic PD-1 of segment (anti-PD-1CH2-CH3) composition Amino acid sequence is SEQ ID No:37, and nucleotides sequence is classified as SEQ ID No:38.

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

On the other hand, the present invention also provides a kind of coding anti-PD-1 and TEM-8 bispecific antibody or its changes Perhaps a kind of expression vector comprising the nucleic acid molecules or one kind include institute to the nucleic acid molecules of body or its functional fragment State the host cell of expression vector.

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

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

In another aspect, the present invention also provides a kind of pharmaceutical compositions comprising the anti-PD-1 and TEM-8 is bis- special Property antibody or its variant or its functional fragment perhaps the nucleic acid molecules perhaps expression vector or host Cell.Preferably, described pharmaceutical composition further includes pharmaceutical 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, as Ali's statin, calicheamicin, Maytansine and times carcinomycin etc.；Immunocyte, such as NK cell, T cell and DC-CIK cell etc..The dosage form of described pharmaceutical composition It can be liquid injection dosage form or solid suppository formulation.

Another aspect, the present invention also provides the anti-PD-1 and TEM-8 bispecific antibody or its variants or its function Energy property segment, the nucleic acid molecules, the expression vector or the host cell are in preparation treating cancer, infectious disease Application in the drug of disease or inflammatory disease.Specifically, wherein the cancer is selected from gastric 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 Column gland cancer, breast cancer, colon cancer, lung cancer, osteocarcinoma, cancer of pancreas, cutaneum carcinoma, head-neck carcinoma, 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, bladder cancer, 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 endocrine system, soft tissue Sarcoma, the neoplasm of central nervous system, primary central nervous system lymphoma, tumor vessel generation, spinal cord axis 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 of solid tumor, lymphocytic lymphoma are a variety of；The infectious diseases be selected from HIV, influenza, bleb, Giardiasis, malaria, leishmaniasis, or the caused infection of following virus: hepatitis virus (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 virus, 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 virus and arboviral encephalitides virus, or infection caused by following bacterium: clothing is former Body, rickettsia, pneumococcus, mycobacteria, staphylococcus, streptococcus, meningococcus, conococci, sand Lei Shi Bacterium, mycetozoan, pseudomonad, salmonella, comma bacillus, corynebacterium diphtheriae, clostridium botulinum, bacillus anthracis, is broken Klebsiella Cold clostridium, Legionella, yersinia pestis, leptospirosis or Lyme disease bacterium, fungus-caused infection below: aspergillus (such as aspergillus fumigatus, aspergillus niger etc.), Candida (such as Candida albicans), candida krusei, Candida glabrata, Candida tropicalis, Cryptococcus neoformans, Blastomyces dermatitidis, Mucoales category (such as mucor, Absidia or rhizopus), Sporotrichum schenckii, Paracoccidioides brasiliensis, thick ball spore bacterium or blooming histoplasma capsulatum, the caused infection of following helminth: Entamoeba historlytica, good fortune Na Shi worm, spine amoeba, Plasmodium vivax, Babesiamicrofti, sucks giardia lamblia, is hidden colon bowel bag worm Sporozoite, Pneumocystis carinii, T. brucei, Cruz trypanosome, Duo Shi Leishmania, 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 syndrome, Idiopathic Thrombocytopenic Purpura, 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.

The present invention also provides the anti-PD-1 and TEM-8 bispecific antibody or its variants or its functional fragment Preparation method comprising the anti-PD-1 and TEM-8 bispecific antibody or its variant or its functional piece can generated Host cell of the present invention is cultivated under conditions of section, and anti-PD-1 and TEM-8 bispecific antibody caused by recycling, Or its variant or its functional fragment.

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

A. can with PD-1 with high specific in conjunction with, block PD-1 and PD-L1 interaction；

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

C. can with TEM-8 with high specific in conjunction with；

D. it is not combined with other vascular endothelial growth factor (such as VEGFR1-3)；

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

The present invention also provides humanization PD-1/TEM-8 bispecific antibody or its variants or its functional fragment.Institute State humanized antibody by be immunized mouse generate source of mouse antibody designed via computer simulation and in conjunction with display technique of bacteriophage and It obtains, and according to the binding characteristic of itself and PD-1 the and TEM-8 ectodomain of different plant species, identifies its corresponding knot Close epitope.The anti-PD-1/TEM-8 bispecific antibody 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 antibody or its variant or its functional fragment.

Bispecific antibody of the present invention or its variant or its functional fragment can be full length antibody, or only include Two antigen-binding portion thereofs, as heavy chain variable region (VH), single-stranded (scFv) or full length antibody are merged with what antigen-binding portion was grouped as Albumen (such as Fig. 2), any form combination is formed by the model that double Specific binding proteins molecules are protected in the present invention as described above In enclosing.

Variant or functional fragment of the present invention include by chemical modification or by that can increase in incorporation liposome Any segment for adding half life, the segment after modification retain binding specificity identical with its derived antibodies and affinity, in this way The new protein molecular with double specific binding properties obtained is also within the protection scope of the present invention.

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

Those skilled in the art can will encode the DNA molecular gram of PD-1/TEM-8 bispecific antibody of the present invention It is grand into carrier, and then convert host cell.Therefore, the present invention also provides for the bispecific antibody shown in Figure 1A-D Recombinant DNA carrier.The variant of PD-1/TEM-8 bispecific antibody and the carrier of functional fragment shown in code pattern 2 and host cell Also within the protection scope of the present 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 for such as pichia pastoris yeast, saccharomyces cerevisiae, fission yeast, trichoderma fungi, such as the insect cell of meadow mythimna separata, The plant cell of such as tobacco, such as bhk cell, Chinese hamster ovary celI, COS cell, myeloma cell mammalian cell.One In a little embodiments, host cell of the present invention is preferably mammalian cell, more preferable bhk cell, Chinese hamster ovary celI, NSO cell or COS cell.

Terms used herein " pharmaceutical composition " indicate to combine at least one to realize certain specific purpose The combination of drug and optional pharmaceutical acceptable carrier, auxiliary material or therapeutic cells.In certain embodiments, the pharmaceutical composition Object includes separated combination on time and/or space, if its can collective effect to achieve the object of the present invention.For example, Ingredient contained by described pharmaceutical composition (such as antibody according to the present invention, nucleic acid molecules, nucleic acid molecules combination and/or sew Close object) object or separate administration can be applied in object with whole.The ingredient contained by the described pharmaceutical composition is dividually When being applied to object, the ingredient 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% glycerol, hyaluronic acid, ethyl alcohol, polyalkylene glycol such as polypropylene glycol or triglycerides etc.. The type of pharmaceutical acceptable carrier used depend particularly on composition according to the present invention whether be formulated as taking orally, nose, intradermal, skin Under, intramuscular or intravenous administration.Composition according to the present invention may include wetting agent, emulsifier or buffer substances as addition Agent.Pharmaceutical composition according to the present invention can be applied by any suitable approach, such as orally available, nose, intradermal, subcutaneous, flesh Interior or intravenous application.

In a related aspect, the present invention provides the PD-1/TEM-8 bispecific antibodies combines with second therapeutic agent Pharmaceutical composition.In one embodiment, the second therapeutic agent be advantageously with PD-1/TEM-8 bispecific antibody Combined any agent.The exemplary agents that can advantageously combine with the PD-1/TEM-8 bispecific antibody include but unlimited In inhibiting other active reagents of PD-1 (including other antibody or its antigen-binding fragment, inhibitor peptides, small molecular antagonists Deng) and/or interference the upstream PD-1 or downstream signal transduction reagent.

The present invention provides the sides that a kind of PD-1/TEM-8 bispecific antibody 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 antibody Meaning treatment method.The example therapy that can advantageously combine with the PD-1/TEM-8 bispecific antibody includes but unlimited In other therapies (including operation, chemotherapy, radiotherapy, cellular immunotherapy etc.) with treatment indication of the present invention.

PD-1/TEM-8 bispecific antibody provided by the invention has high-affinity, a side to PD-1 and TEM-8 simultaneously Face can be specifically bound with the PD-1 of activating T cell, effectively close the combination of PD-1 receptor in PD-L1 albumen and T cell, And then the Immune escaping mechanism of cancer cell caused by PD-1/PD-L access is blocked, activate T cell tumor killing activity；Another party Face can target TEM-8 positive expression tumour, i.e., inhibit swollen in tumor vascular endothelium TEM-8 specific bond with specific expressed Tumor 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 antibody.

Detailed description of the invention

Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:

Fig. 1 is the structural schematic diagram of anti-PD-1/TEM-8 bispecific antibody of the present invention.

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

Fig. 3 is the SDS-PAGE testing result for purifying humanization PD-1, TEM-8 extracellular domain.

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

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

Fig. 6 shows envelope of the anti-PD-1 monoclonal 3,21,45 with 77 pairs of PD-L1 albumen in conjunction with the PD-1 of human T-cell respectively The effect of closing.

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

Fig. 8 shows the agarose gel electrophoresis results of the encoding gene of bispecific antibody of the invention.

The recombinant plasmid map of Fig. 9 code displaying bispecific antibody of the invention.

Figure 10 shows PD-1/TEM-8 bispecific antibody of the invention to the specific bond of PD-1 and TEM-8.

Figure 11 shows PD-1/TEM-8 bispecific antibody of the invention respectively to the PD-1 of PD-L1 albumen and people T cell In conjunction with sealing process.

Figure 12 shows that PD-1/TEM-8 bispecific antibody of the invention acts on the Activation In Vitro of human T-cell.

Figure 13 shows the internal antitumous effect of PD-1/TEM-8 bispecific antibody of the invention.

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

Specific embodiment

The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only For illustrating the present invention, do not limit the scope of the invention in any way.

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

The structure of 1 PD-1/TEM-8 bispecific antibody of embodiment designs

The present invention provides some bispecific antibodies that can specifically bind PD-1 and TEM-8 extracellular domain simultaneously Structural model, the structures of such 4 kinds of principle examples of antibody as shown in Figure 1, in Figure 1A-C the formation of 3 kinds of heterodimers be based on CH3 structural domain mutating technology known to art technology person (knob and hole mutation) (Von Kreudenstein et al., MAbs.2013 5 (5): 646-54).Wherein, the light chain of (A) anti-PD-1 specific antibody is (anti- PD-1 VL-CL)-heavy chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) is (anti-to the light chain with anti-TEM-8 specific antibody PD-1 VL-CL)-heavy chain (anti-PD-1 VH-CH1- hinge area-CH2-CH3) is to the bispecific binding protein of composition；(B) resist The light-heavy chain pair of PD-1 specific antibody and anti-TEM-8 light chain variable region (anti-TEM-8 VL), heavy chain variable region (anti-TEM- 8 VH), hinge area and Fc segment (anti-TEM-8 CH2-CH3) fusion protein composition bispecific binding protein；(C) anti-PD-1 The light-heavy chain pair of light chain variable region, heavy chain variable region, hinge area and Fc fragment fusion protein and anti-TEM-8 specific antibody The bispecific binding protein of composition；(D) heavy chain of the light chain of anti-PD-1 specific antibody and anti-PD-1 specific antibody, with And the bispecific of anti-TEM-8 light chain variable region (anti-TEM-8 VL) and heavy chain variable region (anti-TEM-8 VH) fusion protein composition Binding protein.

The variant of PD-1/TEM-8 bispecific antibody 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 fusion peptide technology known to art technology person (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 heavy chain variable region (anti-PD-1 VH) of CH3 structural domain mutating technology (same to Figure 1A-C) (E) anti-PD-1 known to member and anti- The fusion protein of TEM-8 heavy chain variable region (anti-TEM-8 VH)；(F) single chain variable fragment (scFv) of anti-PD-1 and anti-TEM-8 ScFv composition fusion protein；(G) non-the exempting from of the scFv of the single chain variable fragment (scFv) of anti-PD-1 and anti-TEM-8 composition Epidemic disease Immunoglobulin fusion albumen；(H) anti-PD-1 light chain variable region, heavy chain variable region, hinge area, Fc segment and anti-TEM-8 light chain can Become the tetravalent fusion protein in area, heavy chain variable region composition；(I) anti-PD-1 light chain variable region, heavy chain variable region, hinge area and Fc Fragment fusion protein and anti-TEM-8 light chain variable region, heavy chain variable region, hinge area and Fc fragment fusion protein form double special Property binding protein (note: the function fragment position of specific recognition PD-1 and TEM-8 are interchangeable in figure).

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

The coding gene sequence of source of people PD-1 and TEM-8 extracellular domain is obtained in UniProt database, 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, it is cloned into pcDNA3.1 carrier, utilizes 293T cell (ATCC) protein expression is carried out as host cell, using purification techniques such as affinity chromatography, ion-exchange chromatographies to purpose egg It is white to be purified.

Fig. 3 is source of people PD-1 and TEM-8 extracellular domain protein purification and carries out deglycosylation treated SDS-PAGE Testing result figure.

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

1. immune animal

The purified people PD-1 of 1mg/ml or 0 μ l of TEM-8 extracellular domain protein 10 is complete with isometric Freund respectively Adjuvant at room temperature after evenly mixing, in subcutaneous abdomen multi-point injection to 5 6-8 weeks BALB/C mice bodies.After 7 days, by 1mg/ 0 μ l of ml protein 10 and isometric incomplete Freund's adjuvant at room temperature after evenly mixing, 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 15 μ l of blood, It is stored at room temperature 2h, 10-15min is centrifuged with 25 DEG C, 6000-8000rpm, takes supernatant, detection potency is tested by ELISA.If effect The available i.e. eye of valence detection takes whole blood, is stored at room temperature 2h, is centrifuged 10min with 25 DEG C, 6000-8000 rpm, takes supernatant, and -80 DEG C It saves.If potency is unavailable, continue immune primary.

2. cell fusion

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

3. positive colony screens

Being reacted in screening monoclonal hybridoma using ELISA can secrete in conjunction with PD-1 or TEM-8 extracellular domain egg The clone of Bai Kangti, wherein in conjunction with 4 plants of the preferred clone (clone 3,21,45,77) of PD-1, in conjunction with the preferred clone 3 of TEM-8 Strain (clone 1,5,69), the ELISA testing result of the above clones secrete antibody as illustrated in figures 4 a andb, the clone filtered out The corresponding antigen protein of specific recognition.

Identification of the anti-PD-1 antibody of embodiment 4 in conjunction with other CD28 family proteins

For evaluation and screening go out PD-1 antibody specificity, by member protein PD-1, CD28, CTLA-4 of CD28 family Coating 2h is stood in 37 DEG C of incubators in ELISA Plate with ICOS (R&D Systerm)；4 DEG C of closings are overnight；It is added after washing 100 μ l candidate antibody (1 μ g/ml) are incubated for 1h at 37 DEG C；The goat anti-mouse antibody of diluted HRP label is added after washing 5 times (Abcam), it is incubated at room temperature 1h；100 hole μ l/ of OPD substrate solution is added after washing 5 times, the 2M in 50 holes μ l/ is added after the 5min that develops the color H₂SO₄Terminate reaction.The light absorption value in each hole under 490nm is measured with microplate reader.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.

Enclosed experiment of the PD-1 receptor of the anti-PD-1 antibody on human T cell of embodiment 5 in conjunction with PD-L1

1. the separation of human peripheral T cell

Healthy volunteer's peripheral blood 10mL is extracted, isometric PBS is added into pipe, gently blows and beats into cell suspension 20mL, Cell suspension is slowly added on to the top layer of 20mL LTS1077 lymphocytes separating solution with suction pipe, is centrifuged with 2000 revs/min 20min, sucks the blood plasma of top layer, and draws in the merging centrifuge tube of the monocyte under plasma layer, with PBS washing 3 times, obtains Human peripheral blood mononuclear cell PBMC.

Suspension is made with PBS buffer solution in isolated PBMC, and 100 μ l/ml CD3 antibody (Invitrogen) are added, Featheriness mixes, and is incubated at room temperature 15min；5 μ l/ml immunomagnetic beads (Invitrogen) are added, are incubated for 60min at 4 DEG C, every 10 points Clock concussion is primary；It is rinsed and is diluted with PBS, cell suspension injection is filled in the special test tube of stainless steel wool, separator is put into It is isolated and purified, T cell cannot be purified by separator under high-intensity magnetic field, and Fig. 6 A is the mirror for the T cell purity isolated Determine result.

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

The coding gene sequence of human PD-L 1 extracellular domain is obtained in UniProt database, the side synthesized by full genome Formula obtains encoding gene, and synthetic product is cloned into pEGFP-N1 carrier after EcoR I, Xho I double digestion, utilizes 293T cell (ATCC) carries out protein expression as host cell, utilizes the purification techniques such as affinity chromatography, ion-exchange chromatography Destination protein is purified, the source of people PD-L1 extracellular domain 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 deglycosylation treated SDS-PAGE result.

3. enclosed experiment of the PD-1 receptor of anti-PD-1 antibody on human T cell in conjunction with PD-L1

The antibody (1 μ g/ml) that preferably 4 strain clones generate softly is mixed at room temperature with rh-PD-L1-GFP (1 μ g/ml) Then the human peripheral T cell isolated and purified out is added in mixed protein solution by 30min, clear with PBS after incubation at room temperature 15 minutes It washes 3 times, is analyzed with flow cytometer.

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

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

TEM-8, EFGR, HER2 and VEGFR (R&D Systerm) are stood in 37 DEG C of incubators to coating in ELISA Plate 2h；4 DEG C of closings are overnight；100 μ l candidate antibody (1 μ g/ml) are added after washing, are incubated for 1h at 37 DEG C；It is added after washing 5 times dilute HRP label goat anti-mouse antibody (Abcam) released, is incubated at room temperature 1h；100 hole μ l/ of OPD substrate solution is added after washing 5 times, The 2M H in 50 holes μ l/ is added after colour developing 5min₂SO₄Terminate reaction.The light absorption value in each hole under 490nm is measured with microplate reader.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 respectively and cultivate 3,21,77 and anti-TEM-8 of anti-PD-1 clone clone 1,5,69, culture to cell concentration is 10⁹It is a, Cell is collected by centrifugation, removes cell culture fluid, cell is gently washed twice with the PBS buffer solution (6-8mL) of pre-cooling；With Trizol kit (TAKARA) extracts total serum IgE, with the purity and concentration of UV spectrophotometer measuring RNA；Using total serum IgE as mould Plate, reverse transcription obtain 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 template (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 tumor, 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 template (1 μ L), PCR enzyme (0.5 μ l), PCR water (to 50 μ l).

Amplified production is cloned into pMD19 carrier, carries out recon sequencing after the screening of blue hickie, obtains anti-PD-1 clone 3,21,77 heavy chain and the amino acid sequence of light chain variable region 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.It obtains anti-TEM-8 and clones 1,5,69 Heavy chain and the amino acid sequence of light chain variable region 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.

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

It is utilized according to the variable region amino acid sequence (sequence that embodiment 7 obtains) of the anti-PD-1 of acquisition and TEM-8 antibody IMGT/V-Quest line server analyzes antibody subtype, the CDR region of comprehensive tri- kinds of antibody of Kabat, Chothia and IMGT Numbering scheme determines six CDR region sequences of antibody light chain and heavy chain；Mouse parental antibody Fab is carried out using DS software And its space structure of chimeric humanized antibody Fab, after the molecular model for obtaining chimeric antibody, to parent Fab and chimeric antibody Fab initial model is comprehensively optimized using molecular dynamics simulation, extracts stable average conformation；It is examined using epitope scanning Test the humanized antibody linear epitope that may be present and comformational epitope of design, the Framework Region amino acid site that determination must retain Afterwards, it designs different amino acid mutation sites and obtains humanized antibody.

On this basis, we have obtained multiple humanized antibodies, wherein the highest anti-PD-1 and anti-TEM-8 that scores is anti- Body sequence be respectively clone 81 and clone 76, clone 81 heavy chain and light-chain amino acid sequence be respectively SEQ ID No:25 and 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, and the nucleotide sequence for encoding corresponding amino acid is respectively SEQ ID No:30 and 32.

The building of the anti-PD-1/TEM-8 bispecific antibody expression vector of embodiment 9

Anti- PD-1 (clone 81, preparation method is shown in embodiment 8) in anti-PD-1/TEM-8 bispecific antibody of the 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 The kappa coding nucleotide of coding nucleotide and constant region of light chain CL, and the polypeptide (GGGGS) 3 of link scFv encode Gene is obtained from Life Technologies Inc. (Carlsbad, CA).

Each different chains in the bispecific antibody of 4 kinds of structures shown in Fig. 1, 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 Column design primer (is shown in Table 1).

1 PCR primer sequence of table

PD-1-VH, PD-1-VL, TEM-8-VH, TEM-8-VL, CH1, hinge area (Hinge), Fc, Fc-knob, Fc- The amplification system of hole and CL is 50 μ l, wherein 5 × PCR Buffer (10 μ l), dNTP (4 μ l), primer (2 μ l), cDNA mould Plate (1 μ l), PCR enzyme (0.5 μ l), PCR water (to 50 μ l).Amplification condition are as follows: 98 DEG C are incubated for 3 minutes, and then 25 recycle: 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 is the fine jade of PCR product 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) 3, 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. 1,50 μ l of PCR system, wherein 5 × PCR Buffer (10 μ l), dNTP (4 μ l), draw Object (2 μ l), cDNA template (each 1 μ l of PCR product), PCR enzyme (0.5 μ l), PCR water (to 50 μ l) amplification condition are as follows: 98 DEG C of incubations 3 minutes, then 30 recycled: 98 DEG C are denaturalized 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 is the agarose electrophoresis result figure of PCR product.Each chain coding sequence be SEQ ID No:34,36,38,40, 42,44,46.By building after target fragment digestion to expression vector pcDNA3.1, the recombination of 4 kinds of bispecific antibodies shown in FIG. 1 Plasmid map is shown in Fig. 9 A-G, and wherein Fig. 9 A recombinant plasmid pcDNA3.1-PD-1-LC is that (anti-PD-1 is light by coding PD-1-VL and CL Chain) map；Fig. 9 B recombinant plasmid pcDNA3.1-TEM-8-LC is coding TEM-8-VL and CL (anti-TEM-8 light chain) map；Fig. 9 C Recombinant plasmid pcDNA3.1-PD-1-HC is that coding PD-1-VH, CH1, hinge area (Hinge) and Fc-knob/Fc-hole are (anti- PD-1 heavy chain) map；Fig. 9 D recombinant plasmid pcDNA3.1-TEM-8-HC is coding TEM-8-VH, CH1, hinge area (Hinge) With Fc-knob/Fc-hole (anti-TEM-8 heavy chain) map；Fig. 9 E recombinant plasmid pcDNA3.1-PD-1 scFv-Fc is coding PD- 1 scFv and Fc-knob/Fc-hole map；Fig. 9 F recombinant plasmid pcDNA3.1-TEM-8 scFv-Fc is coding TEM-8 ScFv and Fc-knob/Fc-hole map；Fig. 9 G recombinant plasmid pcDNA3.1-PD-1-HC-TEM-8 scFv is coding PD-1- VH, CH1, hinge area (Hinge), Fc and TEM-8 scFv map.

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

1. utilizing 293F host cell expression bispecific antibody

24 hours before transfection, by 1 × 10⁶293freestyle culture of a 293F cell inoculation in flask In base, 37 DEG C, 8%CO₂, cultivate under the conditions of 130rpm.100 μ l 293fectin are added in the OPtiMEM of 1ml, stirring bar Under part, it is incubated for 5 minutes at room temperature.Meanwhile combining recombinant plasmid by shown in table 2, it is dissolved in 1ml OPtiMEM, DNA is total Amount is 30 μ g.Above-mentioned DNA and 293fectin is thoroughly mixed, and total volume is 2ml, in incubation at room temperature 20 minutes.Then It adds the mixture into cell culture.Cell is cultivated at 37 DEG C, and has 5%CO in incubator₂, trained with 130rpm It supports 5 days.

Each carrier component of 2 rotaring redyeing system of table

Antibody number	Carrier combination
		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, collect supernatant 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 column (being purchased from GE company, 5ml volume), FabAffinity KBPAgarose is affine filler (being purchased from ACROBiosystems company, 5ml volume) and SP cation-exchange chromatography column (are purchased from GE company, 10ml) it is purified, it is operated according to service manual.

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

PD-1, CTLA-4, TEM-8 and VEGFR (R&D Systerm) are stood in 37 DEG C of incubators to coating in ELISA Plate 2h；4 DEG C of closings are overnight；4 kinds of antibody (for sequence referring to embodiment 8, structure is referring to Figure 1A-D) (100 μ g/ after purification are added after washing Ml) gradient dilution liquid (PBS serial dilution (1:102,1:103,1:104,1:105,1:106,1:107,1:108,1: 109), 100 hole μ l/ is incubated for 1h at 37 DEG C；Diluted HRP label goat anti-mouse antibody (Abcam) is added after washing 5 times, It is incubated at room temperature 1h；100 hole μ l/ of OPD substrate solution is added after washing 5 times, the 2M H in 50 holes μ l/ is added after the 5min that develops the color₂SO₄Eventually Only react.The light absorption value in each hole under 490nm is measured with microplate reader.Experimental result is as shown in Figure 10, and 4 kinds of bispecific antibodies can be same When specific recognition PD-1 and TEM-8.

Closing of the anti-PD-1/TEM-8 bispecific antibody of embodiment 12 to the PD-1 receptor of human T-cell in conjunction with PD-L1 Experiment

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

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

By the fresh PBMC bed board for preparing human peripheral into 96 hole flat undersides, after overnight incubation, it is added 10ug/ml's The tetanus toxin (TT) of antibody and 100ng/ml, culture collected supernatant after 3 days, with Life Technology company The IL2 secretion level of Luminex instrument and the CD8+ cytokine detection kits of EMD company detection bispecific antibody and control group (the result is shown in Figure 1 2).The results show that the humanized antibody can stimulate the function of T cell, the PD- of 4 kinds of forms shown in FIG. 1 1/TEM-8 bispecific antibody (sequence is referring to embodiment 8) can effectively activate T cell function, and wherein Fig. 1 D-shaped formula is double special Xenoantibody activation effect is relatively preferable, the secretion of IL-2 caused by the PD-1/TEM-8 bispecific antibody 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%.

The test of the activation T cell function of table 3

Note: P < 0.01 * * compared with the control group.

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

1. different plant tumor model foundation

CT-26 cell in logarithmic growth phase is resuspended in the RPMI 1640 culture medium of serum-free, so that cell concentration It is 1 × 10⁷A cell/ml.By cell infusion in the left shoulder back of mouse, every 200 μ l are denoted 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.It tests the 30th day, swells The volume of tumor has reached 500mm³, mouse is put to death, tumour is separated.

2. anti-PD-1/TEM-8 bispecific antibody kills tumor experiment

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

The dual anti-inhibiting effect result to tumour of table 4

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

Note: P < 0.01 * * compared with the control group.

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

Plantation tumor mouse is divided into 4 groups, every group 4, with the PD-1 monoclonal antibody (clone prepared by embodiment 8 of purifying 81), TEM-8 monoclonal antibody (embodiment 8 prepare clone 76) and anti-PD-1/TEM-8 bispecific antibody (sequence referring to It is embodiment 8, dual anti-shown in Fig. 1 D) in tail vein injection to tumor formation Mice Body, dosage is respectively A group: PBS, B group: 50mg/kg PD-1 monoclonal antibody (clone 81), C group: 50mg/kg TEM-8 monoclonal antibody (clone 76) and D group: the anti-PD-1/TEM-8 of 50mg/kg is bis- special Heterogenetic antibody (dual anti-shown in Fig. 1 D).It injects 1 time every other week, continuous injection 4 times, record mouse interior tumor size becomes Change.The effect that anti-PD-1/TEM-8 bispecific antibody reduces tumour is substantially better than PD-1 monoclonal antibody and TEM-8 monoclonal antibody, mono- in PD-1 Under the action of anti-, TEM-8 monoclonal antibody 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).

Inhibiting effect result of 5 different antibodies of table to tumour

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

Note: P < 0.01 * * compared with the control group.Compared with PD-1 monoclonal antibody group^#P<0.05.Compared with TEM-8 monoclonal antibody group^&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 antibody of embodiment 15

Plantation tumor mouse is divided into 6 groups, every group 4, with the variant of 5 kinds of purifying anti-PD-1/TEM-8 bispecific antibodies (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 at In tumor Mice Body, dosage is respectively A group: PBS, B group: 50mg/kg variant 1 (Fig. 2 E), C group: 50mg/kg variant 2 (Fig. 2 F), D Group: 50mg/kg variant 3 (Fig. 2 G), E group: 50mg/kg variant 4 (Fig. 2 H) and F group: 50mg/kg variant 5 (Fig. 2 I).Every one Week injection 1 time, continuous injection 4 times, records mouse interior tumor size variation.5 kinds of anti-PD-1/TEM-8 bispecific antibodies Variant is likewise supplied with the effect of inhibiting tumour growth, and under the action 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).

Inhibiting effect result of the variant of 6 different antibodies of table to tumour

Note: P < 0.01 * * compared with the control group.

<110>along vast and boundless cellular biological technique (Tianjin) limited liability company

<120>anti-PD-1 and TEM-8 bispecific antibody 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>amino acid sequence of 3 heavy chains is cloned

<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>nucleotide sequence of 3 heavy chain variable regions is cloned

<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>amino acid sequence of 3 light chain variable regions is cloned

<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>nucleotide sequence of 3 light chain variable regions is cloned

<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>amino acid sequence of 21 heavy chain variable regions is cloned

<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>nucleotide sequence of 21 heavy chain variable regions is cloned

<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>amino acid sequence of 21 light chain variable regions is cloned

<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>nucleotide sequence of 21 light chain variable regions is cloned

<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>amino acid sequence of 77 heavy chain variable regions is cloned

<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>nucleotide sequence of 77 heavy chain variable regions is cloned

<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>amino acid sequence of 77 light chain variable regions is cloned

<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>nucleotide sequence of 77 light chain variable regions is cloned

<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>amino acid sequence of 1 heavy chain variable region is cloned

<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>nucleotide sequence of 1 heavy chain variable region is cloned

<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>amino acid sequence of 1 light chain variable region is cloned

<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>nucleotide sequence of 1 light chain variable region is cloned

<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>amino acid sequence of 5 heavy chain variable regions is cloned

<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>nucleotide sequence of 5 heavy chain variable regions is cloned

<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>amino acid sequence of 5 light chain variable regions is cloned

<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>nucleotide sequence of 5 light chain variable regions is cloned

<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>amino acid sequence of 69 heavy chain variable regions is cloned

<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>nucleotide sequence of 69 heavy chain variable regions is cloned

<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>amino acid sequence of 69 light chain variable regions is cloned

<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>nucleotide sequence of 69 light chain variable regions is cloned

<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>amino acid sequence of 81 heavy chain variable regions is cloned

<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>nucleotide sequence of 81 heavy chain variable regions is cloned

<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>amino acid sequence of 81 light chain variable regions is cloned

<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>nucleotide sequence of 81 heavy chain variable regions is cloned

<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>amino acid sequence of 76 heavy chain variable regions is cloned

<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>nucleotide sequence of 76 heavy chain variable regions is cloned

<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>amino acid sequence of 76 light chain variable regions is cloned

<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>nucleotide sequence of 76 light chain variable regions is cloned

<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>amino acid sequence of anti-PD-1 specific antibody 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>nucleotide coding sequence of the heavy chain of anti-PD-1 specific antibody

<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>amino acid sequence of anti-PD-1 specific antibody 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>nucleotide sequence of anti-PD-1 specific antibody 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>the fusion protein amino acid sequence of anti-PD-1 scFv-Fc

<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>nucleic acid sequence of anti-PD-1 scFv-Fc fusion protein

<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>amino acid sequence of anti-TEM8 heavy chain

<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>nucleotide sequence of anti-TEM8 heavy chain

<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>amino acid sequence of anti-TEM8 light chain

<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>nucleotide sequence of anti-TEM8 light chain

<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>amino acid sequence of anti-TEM8 scFv-Fc fusion protein

<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>nucleotide sequence of anti-TEM8 scFv-Fc fusion protein

<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>amino acid sequence of anti-PD-1 heavy chain-TEM8scFv

<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>nucleotide sequence of anti-PD-1 heavy chain-TEM8scFv

<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 (17)

1. a kind of anti-PD-1 and TEM-8 bispecific antibody comprising:

A. the structural domain of specific recognition and combination immune cell surface antigenic PD-1 comprising the weight of anti-PD-1 specific antibody Chain variable region and light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25；Light chain variable region Amino acid sequence as shown in SEQ ID NO:27；And

B. the structural domain of specific recognition and combination tumor endothelial marker TEM-8 comprising the weight of anti-TEM-8 specific antibody Chain variable region and light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:29；Light chain variable region Amino acid sequence as shown in SEQ ID NO:31.

2. anti-PD-1 and TEM-8 bispecific antibody according to claim 1, the specific recognition and combination are immune thin The structural domain of cellular surface antigen PD-1 by sequentially connected anti-PD-1 specific antibody light chain variable region, heavy chain variable region, hinge Sequence and Fc segment composition, or be made of the light chain and heavy chain of one group of anti-PD-1 specific antibody, or special by two groups of anti-PD-1 Property antibody light chain and heavy chain composition, or be made of the light chain variable region and heavy chain variable region of anti-PD-1 specific antibody.

3. anti-PD-1 and TEM-8 bispecific antibody according to claim 1, wherein the specific recognition and combination are swollen Light chain variable region of the structural domain of tumor endothelial marker TEM-8 by sequentially connected anti-TEM-8 specific antibody, weight chain variable Area, hinge area and Fc segment composition, or be made of the light chain and heavy chain of one group of anti-TEM-8 specific antibody, or resisted by two groups The light chain and heavy chain of TEM-8 specific antibody form, or by the light chain variable region and heavy chain variable region of anti-TEM-8 specific antibody Composition.

4. anti-PD-1 and TEM-8 bispecific antibody according to claim 1,

Comprising:

A ' specific recognition and the structural domain for combining immune cell surface antigenic PD-1, by one group of anti-PD-1 specific antibody Light chain and heavy chain composition；And

B ' specific recognition and the structural domain for combining tumor endothelial marker TEM-8, by one group of anti-TEM-8 specific antibody Light chain and heavy chain composition；Alternatively,

It includes

A " specific recognition and the structural domain for combining immune cell surface antigenic PD-1, by one group of anti-PD-1 specific antibody Light chain and heavy chain composition；And

B " specific recognition and the structural domain for combining tumor endothelial marker TEM-8, it is special by sequentially connected anti-TEM-8 Property the light chain variable region of antibody, heavy chain variable region, hinge area and Fc segment composition；Or

It includes

A " ' specific recognition and the structural domain for combining immune cell surface antigenic PD-1, it is special by sequentially connected anti-PD-1 Property the light chain variable region of antibody, heavy chain variable region, hinge area and Fc segment composition；And

B " ' specific recognition and the structural domain for combining tumor endothelial marker TEM-8, by one group of anti-TEM-8 specific antibody Light chain and heavy chain composition；Or

It includes

A " " specific recognition and the structural domain for combining immune cell surface antigenic PD-1, by two groups of anti-PD-1 specific antibodies Light chain and heavy chain composition；And

B " " specific recognition and the structural domain for combining tumor endothelial marker TEM-8, by two groups of anti-TEM-8 specific antibodies Light chain variable region and heavy chain variable region composition.

5. anti-PD-1 and TEM-8 bispecific antibody according to claim 1 or 2, wherein the specific recognition and knot Close the structural domain of immune cell surface antigenic PD-1 and the structure of the specific recognition and combination tumor endothelial marker TEM-8 Domain passes through one or more disulfide bond connections.

6. anti-PD-1 and TEM-8 bispecific antibody according to claim 1, wherein the specific recognition and combination are exempted from The structural domain of the structural domain of epidemic disease cell surface antigen PD-1 and the specific recognition and combination tumor endothelial marker TEM-8 are logical One or more disulfide bond for being located at hinge area are crossed to connect.

7. anti-PD-1 and TEM-8 bispecific antibody according to claim 4, wherein the specific recognition and combination are exempted from The CH3 of the heavy chain of the anti-PD-1 specific antibody of each of the structural domain of epidemic disease cell surface antigen PD-1 respectively with the specificity The heavy chain variable region of the anti-TEM-8 specific antibody of each of identification and the structural domain for combining tumor endothelial marker TEM-8 is logical Cross chemistry key connection.

8. anti-PD-1 and TEM-8 bispecific antibody according to claim 1, be chimeric antibody, humanized antibody or Human antibody.

9. anti-PD-1 and TEM-8 bispecific antibody according to claim 2, wherein the anti-PD-1 specific antibody The amino acid sequence of heavy chain is SEQ ID NO:33, and nucleotide coding sequence is SEQ ID NO:34；The anti-PD-1 specificity The amino acid sequence of the light chain of antibody is SEQ ID NO:35, and nucleotide coding sequence is SEQ ID NO:36；It is described by successively Light chain variable region, heavy chain variable region, the specificity of hinge area and Fc segment composition of the anti-PD-1 specific antibody of connection Identification and the amino acid sequence for combining the structural domain of immune cell surface antigenic PD-1 are SEQ ID NO:37, nucleotide coding sequence It is classified as SEQ ID NO:38.

10. anti-PD-1 and TEM-8 bispecific antibody according to claim 3, wherein the anti-TEM-8 specific antibody Heavy chain amino acid sequence be SEQ ID NO:39, nucleotide coding sequence be SEQ ID NO:40；The anti-TEM-8 is special Property antibody light chain amino acid sequence be SEQ ID NO:41, nucleotide coding sequence be SEQ ID NO:42；It is described by according to Light chain variable region, heavy chain variable region, hinge area and the Fc segment of the anti-TEM-8 specific antibody of secondary connection form described special Property identification and combine tumor endothelial marker TEM-8 structural domain amino acid sequence be SEQ ID NO:43, nucleotide coding Sequence is SEQ ID NO:44.

11. anti-PD-1 and TEM-8 bispecific antibody according to claim 1, wherein the specific recognition and combination The nucleotide coding sequence of heavy chain variable region in the structural domain of PD-1 is as shown in SEQ ID NO:26；The nucleosides of light chain variable region Coding sequences are as shown in SEQ ID NO:28.

12. anti-PD-1 and TEM-8 bispecific antibody according to claim 1, wherein the specific recognition and combination The nucleotide coding sequence of heavy chain variable region in the structural domain of TEM-8 is as shown in SEQ ID NO:30；The core of light chain variable region Thuja acid coded sequence is as shown in SEQ ID NO:32.

13. a kind of nucleic acid for encoding anti-PD-1 and TEM-8 bispecific antibody according to any one of claim 1 to 12 Molecule.

14. a kind of expression vector comprising nucleic acid molecules according to claim 13.

15. a kind of host cell comprising expression vector according to claim 14.

16. a kind of pharmaceutical composition comprising anti-PD-1 and TEM-8 according to any one of claim 1 to 12 is bis- special Heterogenetic antibody.

17. pharmaceutical composition according to claim 16, wherein described pharmaceutical composition includes medicinal carrier and/or auxiliary Material.