Pharmaceutical compositions containing anti-TIGIT antibodies and anti-PD-1-anti-VEGFA bispecific antibodies and uses thereof

Abstract

The present invention belongs to the field of medicine and relates to an anti-TIGIT antibody, a pharmaceutical composition and use thereof, in particular the use in combination with an anti-PD-1-anti-VEGFA antibody for treating and / or preventing tumors. Specifically, the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, in which the heavy chain variable region of the antibody comprises HCDR1 to HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 3 to 5, respectively, and the light chain variable region of the antibody comprises LCDR1 to LCDR3 whose amino acid sequences are shown in SEQ ID NOs: 8 to 10, respectively. The antibody of the present invention can effectively bind to TIGIT and may be used for preventing and treating tumors.

Description

[Technical field]

[0001] The present invention belongs to the pharmaceutical field and relates to an anti-TIGIT antibody, its pharmaceutical composition and use. Specifically, the present invention relates to an anti-TIGIT monoclonal antibody. [Background technology]

[0002] TIGIT (also called T cell Ig and ITIM domain, or WUCAM, Vstm3, VSIG9) is a member of the poliovirus receptor (PVR) / Nectin family. TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type I transmembrane domain, and an intracellular domain with canonical immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoglobulin tyrosine-based tail (ITT) motifs. TIGIT is highly expressed in lymphocytes, particularly in effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells, and natural killer (NK) cells (Yu X, Harden K, Gonzalez LC, et al. The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells[J]. Nature immunology, 2009, 10(1): 48).

[0003] CD155 (also called PVR, Necl5 or Tage4), CD112 (also called PVRL2 / nectin 2) and CD113 (also called PVRL3) are ligands bound by TIGIT (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors[J]. Nature Reviews Immunology, 2015, 15(4): 243-254), of which CD155 is a high affinity ligand for TIGIT. In NK cells, TIGIT binds to the ligands CD155 and CD112, which can suppress the killing activity of NK cells against TIGIT-high expressing cells (Stanietsky N, Simic H, Arapovic J, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity[J]. Proceedings of the National Academy of Sciences, 2009, 106(42): 17858~17863). It has been reported that simultaneous blockade of PD-1 and TIGIT can increase the killing activity of CD8+ T cells (Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function[J]. Cancer cell, 2014, 26(6): 923~937).Recent research has demonstrated that TIGIT acts as an immune checkpoint for NK cells, and that during tumor growth, the inhibitory receptor TIGIT leads to NK cell exhaustion. Anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and are used in immunotherapy of various tumors (Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J]. Nature immunology, 2018, 19(7): 723~732).

[0004] Other reports have shown that TIGIT blockade alone or in combination with PD-1 and CD96 blockade significantly reduces the growth of B16 melanoma in wild-type and Cd155- / - mouse models (Li XY, Das I, Lepletier A, et al.Cd155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms.J Clin Invest 2018; 128: 2613-25). CD112R blockade alone or in combination with TIGIT and / or PD-1 blockade can increase the cytokine production of TILs in ovarian, endometrial and lung tumors (Whelan S, Ophir E, Kotturi MF, et al.PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8 + T-cell Function. Cancer Immunol Res2019; 7: 257-68).

[0005] Anti-TIGIT antibody drugs are new immune checkpoint antibody drugs with a wide range of potential applications and are used in tumor immunotherapy. Tiragolumab, developed by Roche, has already undergone Phase 3 clinical trials, and it has been reported that the combination of TIGIT monoclonal antibody Tiragolumab and PD-L1 drug Tecentriq (atezolizumab) as first-line treatment is effective in treating PD-L1-positive metastatic non-small cell lung cancer (NSCLC). In a Phase 2 clinical trial of patients, the combination of Tiragolumab and Tecentriq was well tolerated and reduced the risk of disease progression by 43%, demonstrating a significant combination effect (Exit C. Roche to present first clinical data on novel anti-TIGIT cancer immunotherapy tiragolumab at ASCO[J]). According to existing clinical information records, TIGIT is an important target for the treatment of non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, B lymphocytic tumors, and plasma cell carcinomas.

[0006] However, existing anti-human TIGIT antibody drugs have low affinity, and anti-TIGIT antibodies with high affinity are still lacking. Therefore, it is of great significance to develop antibody drugs for the treatment of autoimmune diseases that have high affinity for TIGIT, thereby improving the therapeutic effect and reducing toxicity and side effects.

[0007] The transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed on activated T cells, B cells, and myeloid cells. The PD-1 ligands PDL1 (also abbreviated as PD-L1) and PDL2 (also abbreviated as PD-L2) belong to the B7 superfamily, of which PDL1 is expressed on many types of cells, including T cells, B cells, and endothelial and epithelial cells, whereas PDL2 is expressed only on antigen-presenting cells such as dendritic cells and macrophages.

[0008] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune evasion. PD-1 is mainly expressed in immune cells such as T cells, while its ligand PDL1 is mainly highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway activates suppressed T cells, which can then attack cancer cells. Blocking the PD-1 / PDL1 signaling pathway can promote the proliferation of tumor antigen-specific T cells, exert tumor cell killing effects, and ultimately suppress local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455~2465). In addition, tumors that highly express PDL1 are associated with cancers that are difficult to detect (Hamanishi et al, 2007, Proc. Natl. Acad. Sci. USA 104: 3360-5). An effective implementation method is to inject anti-PD-1 antibodies into the body to control the expression of PD-1. Antibodies against the PD-1 pathway are generally believed in the industry to provide breakthrough advances in the treatment of many types of tumors, including those used to treat non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol. 42(3): 466-473), leukemia and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets. 13(7): 768-74), due to the broad anti-tumor potential and surprising efficacy of PD-1 antibodies.

[0009] Vascular endothelial growth factor (VEGF) is a growth factor that promotes the division and proliferation of endothelial cells, promotes the formation of new blood vessels, and enhances vascular permeability. It exerts its function by binding to vascular endothelial growth factor receptors on the cell surface and activating the tyrosine kinase signaling pathway. In tumor tissues, tumor cells, tumor-infiltrating macrophages, and mast cells secrete high levels of VEGF, which stimulates tumor vascular endothelial cells in a paracrine manner, promotes the proliferation and migration of endothelial cells, induces angiogenesis, promotes the sustained growth of tumors, and also enhances vascular permeability, causes fibrin deposition in surrounding tissues, promotes the infiltration of monocytes and fibroblast endothelial cells, promotes the formation of tumor matrix and the entry of tumor cells into new blood vessels, and promotes tumor metastasis. Therefore, inhibition of tumor angiogenesis is currently considered to be one of the most promising tumor treatment methods. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4) and Neuropilin-1 (NRP-1). The first three of these receptors are structurally similar and all belong to the tyrosine kinase superfamily. All of them are composed of three parts: an extramembrane region, a transmembrane segment, and an intramembrane region, of which the extramembrane region is composed of an immunoglobulin-like domain, and the intramembrane region belongs to the tyrosine kinase region. VEGFR1 and VEGFR2 are mainly located on the surface of vascular endothelial cells, and VEGFR3 is mainly located on the surface of lymphatic endothelial cells.

[0010] VEGF family molecules have different affinities for several receptors. VEGFA mainly binds to VEGFR1, VEGFR2 and NRP-1 to exert its action. VEGFR1 is the earliest receptor discovered, and under normal physiological conditions, the affinity between VEGFR1 and VEGFA is higher than that between VEGFR2 and VEGFA, but its intracellular tyrosinase activity is lower than that of VEGFR2 (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5),(2016):146~148).

[0011] VEGFR2 is a major regulator of angiogenesis and construction, and VEGFR2 has a much higher tyrosine kinase activity than VEGFR1. VEGFR2 mediates the behavior of vascular endothelial cells, such as proliferation and differentiation, as well as the process of angiogenesis and vascular permeability after binding to the ligand VEGFA (Roskoski R Jr. et al., Crit Rev Oncol Hematol, 62(3),(2007):179-213), and VEGFA mediates the transcriptional expression of intracellular related protein genes via the downstream PLC-γ-PKC-Raf-MEK-MAPK signaling pathway after binding to VEGFR2, promoting the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13),(1999):2221-2230).

[0012] VEGFR3 is a member of the tyrosine kinase family and is mainly expressed in vascular endothelial cells during the embryonic stage and lymphatic endothelial cells during adulthood. VEGFC and VEGFD bind to VEGFR3 to stimulate the proliferation and migration of lymphatic endothelial cells and promote lymphangiogenesis. NRP-1 is a non-tyrosine kinase transmembrane protein that cannot transmit biological signals independently, but can mediate signal transduction after forming a complex with the VEGF tyrosine kinase receptor. (Ma Li, Chinese Journal of Eugenics and Genetics, 24(5), (2016):146~148).

[0013] VEGFA and VEGFR2 are mainly involved in the control of angiogenesis, and before and after the binding of VEGFA with VEGFR2, multiple intermediate signals in the upstream and downstream pathways form cascade reactions, ultimately changing the physiological functions of endothelial cells in different ways, such as proliferation, survival, migration, increased permeability, and invasion into surrounding tissues (Dong Hongchao et al., Modern Oncology, Vol. 22, Issue 9, September 2014, pp. 2231-3).

[0014] Currently, many humanized monoclonal antibodies targeting human VEGF, especially VEGFA, such as bevacizumab, have been approved by the US Food and Drug Administration in 2004 for the treatment of various types of tumors, including non-small cell lung cancer, renal cell carcinoma, cervical cancer, and metastatic colorectal cancer. Therefore, the development of more effective therapeutic approaches and combination dosing regimens is of great clinical significance. Summary of the Invention

[0015] Through intensive research and creative work, the inventors have used a mammalian cell expression system to express recombinant human TIGIT as an antigen, immunized mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells. Through screening of a large number of samples, the inventors obtained a hybridoma cell line LT019 (deposit number is CCTCC NO: C2020208).

[0016] The present inventors have surprisingly found that the hybridoma cell line LT019 can secrete and produce a specific monoclonal antibody (named 26B12) that specifically binds to human TIGIT, and that the monoclonal antibody can bind to TIGIT very effectively, reduce the immune cell suppression effect of TIGIT, promote T cell activity, reverse NK cell depletion, and enhance the killing effect of immune cells against tumors.Furthermore, the present inventors have creatively produced humanized antibodies of anti-human TIGIT (named 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4).

[0017] The present inventors further surprisingly discovered that the antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 of the present invention have the activity of binding to TIGIT and have very strong affinity, and 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 can effectively reduce the activity of TIGIT.

[0018] Furthermore, the present inventors discovered that combined use of an anti-TIGIT antibody and an anti-PD-1-anti-VEGFA bispecific antibody is effective in preventing and treating tumors.

[0019] The antibodies of the present invention may be used for the treatment and / or prevention of diseases such as tumors (e.g., non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, plasma cell carcinoma), etc. To this end, the following inventions are provided.

[0020] One aspect of the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, comprising: the anti-TIGIT antibody comprises HCDR1 to HCDR3 contained in a heavy chain variable region represented by SEQ ID NO: 1 and LCDR1 to LCDR3 contained in a light chain variable region represented by SEQ ID NO: 6; Preferably, according to the IMGT numbering system, the heavy chain variable region of the above antibody comprises HCDR1 to HCDR3 whose amino acid sequences are set forth in SEQ ID NO:3 to SEQ ID NO:5, respectively, and the light chain variable region of the above antibody comprises LCDR1 to LCDR3 whose amino acid sequences are set forth in SEQ ID NO:8 to SEQ ID NO:10, respectively. In one or more embodiments of the invention, the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17; and The amino acid sequence of the light chain variable region of the above antibody is selected from SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25. In one or more embodiments of the invention, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the above antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the above antibody is shown in SEQ ID NO: 19; The amino acid sequence of the heavy chain variable region of the above antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the above antibody is shown in SEQ ID NO: 19; The amino acid sequence of the heavy chain variable region of the above antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the above antibody is set forth in SEQ ID NO: 21; The amino acid sequence of the heavy chain variable region of the above antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the above antibody is set forth in SEQ ID NO: 23; The amino acid sequence of the heavy chain variable region of the above antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the above antibody is set forth in SEQ ID NO: 21; The amino acid sequence of the heavy chain variable region of the above antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the above antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 25; or The amino acid sequence of the heavy chain variable region of the above antibody is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region of the above antibody is shown in SEQ ID NO:25. In one or more embodiments of the invention, the antibody comprises non-CDR regions, and the non-CDR regions are derived from a non-murine species, such as a human antibody.

[0021] In one or more embodiments of the present invention, the heavy chain constant region of the antibody is an Ig gamma-1 chain C region (e.g., NCBI accession number: P01857), and the light chain constant region is an Ig kappa chain C region (e.g., NCBI accession number: P01834).

[0022] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody, chimeric antibody, or bibody.

[0023] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof has a K of less than 4E-10 or less than 4E-11. D and preferably binds to TIGIT-mFc at the above K D is measured on a Fortebio molecular interaction instrument.

[0024] In one or more embodiments of the invention, the anti-TIGIT antibody or antigen-binding fragment thereof, wherein the antibody has an EC 50 and preferably binds to TIGIT at the EC 50 is measured by a flow cytometer.

[0025] In some embodiments of the present invention, the anti-TIGIT antibody is a monoclonal antibody.

[0026] In some embodiments of the present invention, the anti-TIGIT antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (eg, a bispecific antibody).

[0027] In some embodiments of the invention, the antigen-binding fragment is selected from a Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, a complementarity determining region fragment, a single chain antibody (e.g., scFv), a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0028] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment is an antibody produced by hybridoma cell line LT019, which is deposited at the China Center for Typical Culture Collection (CCTCC), Wuhan, China, zip code 430072, with deposit number CCTCC NO: C2020208.

[0029] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding an anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the present invention.

[0030] A further aspect of the present invention pertains to a vector comprising the isolated nucleic acid molecule of the present invention.

[0031] A further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention or a vector of the invention.

[0032] A further aspect of the present invention relates to the hybridoma cell line LT019, which has been deposited at the China Typical Culture Collection Center (CCTCC) and has the deposit number CTCCC NO: C2020208.

[0033] A further aspect of the present invention relates to a complex comprising an antibody and a coupling moiety, wherein the antibody is an anti-TIGIT antibody or an antigen-binding fragment thereof described in any one of the claims of the present invention, and the coupling moiety is a detectable label, preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0034] A further aspect of the present invention relates to a kit comprising an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of the invention or a complex of the invention, Preferably, the kit further comprises a second antibody that specifically identifies the antibody, and optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0035] A further aspect of the invention relates to the use of an antibody according to any one of the invention or a complex of the invention in the preparation of a kit, said kit being used for detecting the presence or level of TIGIT in a sample.

[0036] A further aspect of the present invention relates to a pharmaceutical composition comprising an anti-TIGIT antibody or an antigen-binding fragment thereof described in any one of the present invention or a complex of the present invention, optionally further comprising a pharma- ceutically acceptable vector and / or excipient.

[0037] In one or more embodiments of the invention, the pharmaceutical composition further comprises one or more anti-PD-1 antibodies or anti-VEGF antibodies.

[0038] In one or more embodiments of the present invention, the pharmaceutical composition has a mass ratio of the anti-TIGIT antibody or antigen-binding fragment thereof to the anti-PD-1 antibody or anti-VEGF antibody, calculated based on the mass of the antibodies, of (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1.

[0039] A further aspect of the invention relates to a combination product (e.g. a kit) comprising a first product and a second product, packaged separately, wherein: The first product comprises an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of the present invention, a conjugate of the present invention, or a pharmaceutical composition according to any one of the present invention, the second article comprises at least one anti-PD-1 antibody or one anti-VEGF antibody, such as an anti-PD-1-anti-VEGF A bispecific antibody; Preferably, the combination product further comprises a separately packaged third product comprising one or more chemotherapeutic agents; Preferably, the first product and the second product further independently comprise one or more pharma- ceutically acceptable adjuvants; Preferably, the combination product further comprises product instructions.

[0040] In one or more embodiments of the present invention, the above combination product has a mass ratio of the anti-TIGIT antibody or antigen-binding fragment thereof to the anti-PD-1 antibody or anti-VEGF antibody, calculated based on the mass of the antibodies, of (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.

[0041] In one or more embodiments of the invention, the anti-PD-1 antibody or anti-VEGF antibody is an anti-PD-1-anti-VEGFA bispecific antibody.

[0042] A further aspect of the invention relates to the use of an antibody according to any one of the invention, a conjugate according to the invention, a pharmaceutical composition according to any one of the invention or a combination product according to any one of the invention in the preparation of a medicament for treating and / or preventing a tumor, preferably said tumor being selected from one or more of non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma and plasma cell carcinoma.

[0043] The antibody according to any one of the invention, the conjugate according to the invention, the pharmaceutical composition according to any one of the invention or the combination product according to any one of the invention are used for treating and / or preventing tumors, preferably said tumors are selected from one or more of: non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma and plasma cell carcinoma.

[0044] A further aspect of the invention relates to a method for treating and / or preventing a tumor, comprising the step of administering to a subject in need thereof an effective amount of an antibody according to any one of the invention, a conjugate according to the invention, a pharmaceutical composition according to any one of the invention or a combination product according to any one of the invention, preferably said tumor is selected from one or more of: non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma and plasma cell carcinoma.

[0045] The present invention relates to a method for preventing and / or treating a tumor (particularly a malignant tumor), comprising administering to a subject a therapeutically effective amount of an anti-TIGIT antibody, co-administering an anti-PD-1-anti-VEGFA bispecific antibody, and more preferably administering to the subject one or more drugs for treating the tumor (preferably, said drugs are selected from the group consisting of chemotherapeutic agents or growth inhibitors, targeted therapeutic agents, antibody-drug conjugates, T cells expressing a chimeric antigen receptor, antibody or antigen binding fragments, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and combinations thereof, alkylating agents, antimetabolites, and further co-administering an antitumor drug such as an anti-PD-1, anti-VEGFA bispecific antibody, anti-TIGIT antibody, anti-PD-1-anti-VEGFA bispecific antibody, or anti-tumor chemotherapeutic agent, preferably a platinum-based drug such as cyclophosphamide, pemetrexed, cisplatin, carboplatin, oxaliplatin, adriamycin, paclitaxel, vinblastine, tamoxifen, megestrol, goserelin, asparaginase, or fluorouracil, preferably, the anti-TIGIT antibody, anti-PD-1-anti-VEGFA bispecific antibody, and the tumor chemotherapeutic agent are administered simultaneously or sequentially.

[0046] In one or more embodiments of the invention, the chemotherapeutic agent or growth inhibitory agent is selected from alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, anti-microtubule agents, EGFR antagonists, VEGF antagonists, angiopoietin 2 antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0047] In one or more embodiments of the invention, the targeted therapeutic agent is selected from a B-raf inhibitor, a MEK inhibitor, a K-ras inhibitor, a c-Met inhibitor, an Alk inhibitor, a phosphatidylinositol 3-kinase inhibitor, an Akt inhibitor, an mTOR inhibitor, a VEGF inhibitor, a bisphosphatidylinositol 3-kinase / mTOR inhibitor, and combinations thereof.

[0048] In one or more embodiments of the invention, the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethylauristatin E, calicheamicin, esperamicin, and a radioisotope chelator.

[0049] In one or more embodiments of the invention, the tumor is: Cervical cancer (e.g., metastatic cervical cancer), endometrial cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer (e.g., squamous non-small cell lung cancer and non-squamous non-small cell lung cancer), pharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, gastric cancer (e.g., advanced gastric cancer, gastrointestinal cancer, gastric adenocarcinoma or gastroesophageal junction adenocarcinoma), liver cancer (e.g., hepatocellular carcinoma), intestinal cancer, rectal cancer, colon cancer, colorectal cancer Cancer, bile duct cancer, hepatic bile cancer, biliary tract cancer, cholangiocarcinoma, pancreatic cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), ovarian cancer (e.g., advanced ovarian cancer), fallopian tube cancer, peritoneal cancer, glioma (e.g., glioma, recurrent glioma), skin cancer, melanoma, leukemia (e.g., acute myeloid leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma), plasma cell cancer, bone cancer, sarcoma, osteomyelitis In one embodiment, the cancer is selected from one or more of the following: myeloma, chondrosarcoma, neuroblastoma, myeloma (e.g., multiple myeloma), large cell neuroendocrine carcinoma, urothelial carcinoma (e.g., upper tract urothelial carcinoma or bladder carcinoma), prostate cancer, testicular cancer, peripheral T-cell lymphoma, nasopharyngeal carcinoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, head and neck cancer, brain cancer (e.g., invasive brain cancer, e.g., glioblastoma), squamous cell carcinoma, basal cell carcinoma, adenoma, breast cancer (e.g., trigeminal breast cancer), thymic carcinoma, ileocecal adenocarcinoma, urinary adenocarcinoma, mucinous or serous cystadenocarcinoma, leiomyosarcoma, rhabdomyosarcoma, chorioepithelial carcinoma, malignant vivisection, malignant Sertoli stromal cell tumor, malignant granular cell tumor, anaplastic cell tumor, glioblastoma, mycosis, Merkel cell carcinoma, and other hematological malignancies.

[0050] In one or more embodiments of the invention, the anti-PD-1 bispecific antibody comprises: a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; Wherein, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin; Among them, the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 contained in the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 31 (preferably, HCDR1 to HCDR3 contained in SEQ ID NO: 35 to 37, respectively, according to the IMGT numbering system); and the light chain variable region comprises LCDR1 to LCDR3 contained in the light chain variable region whose amino acid sequence is shown in SEQ ID NO: 33 (preferably, LCDR1 to LCDR3 contained in SEQ ID NO: 38 to 40, respectively, according to the IMGT numbering system); the heavy chain variable region of the single chain antibody comprises HCDR1 to HCDR3 contained in the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 41 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 45 to 47, respectively, according to the IMGT numbering system), and the light chain variable region comprises amino acid sequence 43 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 48 to 50, respectively, according to the IMGT numbering system); Or, the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 contained in the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 41 (preferably, HCDR1 to HCDR3 contained in SEQ ID NO: 45 to 47, respectively, according to the IMGT numbering system); and the light chain variable region comprises LCDR1 to LCDR3 contained in the light chain variable region whose amino acid sequence is shown in SEQ ID NO: 43 (preferably, LCDR1 to LCDR3 contained in SEQ ID NO: 48 to 50, respectively, according to the IMGT numbering system); the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 contained in the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 31 (preferably, HCDR1 to HCDR3 contained in SEQ ID NO: 35 to 37, respectively, according to the IMGT numbering system), and the light chain variable region comprises LCDR1 to LCDR3 contained in the light chain variable region whose amino acid sequence is shown in SEQ ID NO: 33 (preferably, LCDR1 to LCDR3 contained in SEQ ID NO: 38 to 40, respectively, according to the IMGT numbering system); The immunoglobulin is of the human IgG1 subtype.

[0051] In one or more embodiments of the invention, the bispecific antibody, wherein the heavy chain constant region of the immunoglobulin comprises the following mutations according to the EU numbering system: L234A and L235A, or L234A and G237A, or L235A and G237A, or Has L234A, L235A, and G237A.

[0052] In the present invention, unless otherwise specified, the alphabet before the site indicates the amino acid before mutation, and the alphabet after the site indicates the amino acid after mutation.

[0053] In one or more embodiments of the invention, the bispecific antibody, wherein the heavy chain constant region of the immunoglobulin is The gene further comprises one or more mutations selected from the group consisting of N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A.

[0054] In one or more embodiments of the invention, the bispecific antibody is The amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 33, and the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 43.

[0055] In one or more embodiments of the invention, the bispecific antibody comprises: The amino acid sequence of the heavy chain of said immunoglobulin is shown in SEQ ID NO:27, and the amino acid sequence of the light chain is shown in SEQ ID NO:29.

[0056] In some embodiments of the present invention, the single chain antibody in the bispecific antibody is linked to the C-terminus of the heavy chain of an immunoglobulin. Since an immunoglobulin has two heavy chains, two single chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single chain antibody molecules are the same.

[0057] In some embodiments of the present invention, the bispecific antibody has two single-chain antibodies, and one end of each single-chain antibody is linked to the C-terminus or N-terminus of two heavy chains of an immunoglobulin, respectively.

[0058] In some embodiments of the invention, the V H and V L There is a disulfide bond between the V H and V LMethods for introducing a disulfide bond between the carboxyl group and the carboxyl group are well known in the art, and are described in, for example, U.S. Pat. No. 5,747,654; Rajagopal et al., Prot. Engin. 10(1997)1453-1459; Reiter et al., Nat. Biotechnol. 14(1996)1239-1245; Reiter et al., Protein Engineering 8(1995):1323-1331; Webber et al., Molecular Immunology 32(1995):249-258; Reiter et al., Immunity 2(1995)281-287; Reiter et al., JBC 269(1994):18327-18331; Reiter et al., Inter. J. of Cancer 58 (1994) 142-149, or Reiter et. al., Cancer Res. 54 (1994) 2714-2718, which are incorporated herein by reference.

[0059] In one or more embodiments of the invention, the first protein functional region in the bispecific antibody is linked directly or via a linking fragment to the second protein functional region and / or the heavy chain variable region of the single chain antibody is linked directly or via a linking fragment to the light chain variable region of the single chain antibody.

[0060] In one or more embodiments of the invention, the linked fragment in the bispecific antibody is (GGGGS)n, where n is a positive integer, and preferably, n is 1, 2, 3, 4, 5 or 6.

[0061] In one or more embodiments of the invention, the first protein functional region and the second protein functional region in the bispecific antibody are independently one, two or more than two.

[0062] In one or more embodiments of the invention, the single chain antibody in the bispecific antibody is linked to the C-terminus of an immunoglobulin heavy chain.

[0063] In one or more embodiments of the invention, the first protein functional region is linked to the second protein functional region via a first linking fragment, and the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody via a second linking fragment, and the first linking fragment and the second linking fragment are homologous or different; Preferably, the amino acid sequences of the first linked fragment and the second linked fragment are independently selected from SEQ ID NO: 52 and SEQ ID NO: 53; Preferably, the amino acid sequences of the first linked fragment and the second linked fragment are both shown in SEQ ID NO:53.

[0064] In one or more embodiments of the invention, the bispecific antibody is a monoclonal antibody.

[0065] In one or more embodiments of the invention, the bispecific antibody is a humanized antibody.

[0066] Another aspect of the present invention relates to a unit preparation, preferably for treating a tumor, wherein the unit preparation comprises 1 mg to 10,000 mg (preferably 10 mg to 1,000 mg, more preferably 50 mg to 500 mg, 100 mg to 400 mg, 150 mg to 300 mg, 150 mg to 250 mg, or 200 mg) of an anti-TIGIT antibody according to any aspect of the present invention and 1 mg to 10,000 mg (preferably 1 mg to 1,000 mg, more preferably 50 mg to 500 mg, 100 mg to 400 mg, 150 mg to 300 mg, 150 mg to 250 mg, 200 mg, or 100 mg) of an anti-TIGIT antibody according to any aspect of the present invention. mg) of the anti-PD-1-anti-VEGFA bispecific antibody according to any aspect of the present invention, and optionally one or more drugs for treating a tumor according to the present invention (e.g., chemotherapeutic drugs such as platinum-based drugs and / or fluorouracil-based antitumor drugs), wherein the anti-TIGIT antibody, the anti-PD-1-anti-VEGFA bispecific antibody, and the drug for treating a tumor are each packaged separately.

[0067] The present invention relates to a method for preventing or treating cancer or tumor, comprising administering to a subject in need thereof one or more unit formulations according to the present invention, wherein the anti-PD-1-anti-VEGFA bispecific antibody, the anti-TIGIT antibody and the drug for treating tumor in the unit formulation are each administered separately.

[0068] In another embodiment of the present invention, it relates to a single drug dosage unit, preferably used for treating a tumor, which comprises 0.1 mg to 10000 mg (preferably 1 mg to 1000 mg, more preferably 50 mg to 500 mg, 100 mg to 400 mg, 150 mg to 300 mg, 150 mg to 250 mg, 200 mg or 100 mg) of an anti-TIGIT antibody according to any one of the present invention, and 0.1 mg to 10000 mg (preferably 1 mg to 1000 mg, more preferably 50 mg to 500 mg, 100 mg to 400 mg, 150 mg to 300 mg, 150 mg to 250 mg, 200 mg or 100 mg) of an anti-PD-1-anti-VEGFA bispecific antibody according to any one of the present invention.

[0069] In one or more embodiments of the present invention, the anti-TIGIT antibody, the anti-PD-1-anti-VEGFA bispecific antibody and / or the medicament for treating a tumor are in a form suitable for intravenous injection or intravenous infusion, preferably in liquid form.

[0070] In one or more embodiments of the invention, the step of administering to a subject an effective amount of an anti-TIGIT antibody described in any one of the claims of the invention and / or an anti-PD-1-anti-VEGFA bispecific antibody described in any one of the claims of the invention is before or after surgical treatment, and / or before or after radiation therapy.

[0071] In one or more embodiments of the present invention, the single dose of the anti-TIGIT antibody of any one of the present invention and / or the anti-PD-1-anti-VEGFA bispecific antibody of any one of the present invention is 0.1 mg to 100 mg per kg of body weight, and preferably 1 mg to 10 mg; alternatively, the single dose of the anti-TIGIT antibody of any one of the present invention and / or the anti-PD-1-anti-VEGFA bispecific antibody of any one of the present invention is 10 mg to 1000 mg per subject, and preferably 50 mg to 500 mg, 100 mg to 400 mg, 150 mg to 300 mg, 150 mg to 250 mg, or 200 mg; Preferably, the drug is administered twice a day to about once every two days, or once every three days, once every four days, once every five days, once every six days, once every ten days, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks. Preferably, the mode of administration is intravenous infusion or intravenous injection.

[0072] The light and heavy chain variable regions determine antigen binding, and each chain variable region contains three hypervariable regions, called complementarity determining regions (CDRs) (heavy (H) chain CDRs contain HCDR1, HCDR2, and HCDR3, and light (L) chain CDRs contain LCDR1, LCDR2, and LCDR3, as named by Kabat et al., Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3: 91-3242). There are currently several ways to determine antibody CDR regions, given an antibody heavy and light chain variable region sequence, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, application of each of the definitions of CDRs to an antibody or variants thereof is within the scope of the term as defined and used herein. Given the variable region amino acid sequence of an antibody of interest, one of skill in the art can generally determine particular CDRs without reliance on any experimental data other than the sequence itself.

[0073] Preferably, the CDRs may be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0074] By technical means well known to those skilled in the art, the amino acid sequence of the CDR region of the monoclonal antibody sequence is analyzed based on the IMGT definition, for example, in the VBASE2 database.

[0075] The antibodies 26B12, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 of the present invention have the same CDRs.

[0076] The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GHSFTSDYA (SEQ ID NO: 3) HCDR2: ISYSDST (SEQ ID NO: 4) HCDR3: ARLDYGNYGGAMDY (SEQ ID NO:5), The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QHVSTA (SEQ ID NO: 8) LCDR2: SAS (SEQ ID NO: 9) LCDR3: QQHYITPWT (sequence number 10).

[0077] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratory procedures used herein are common procedures widely used in the art. At the same time, in order to better understand the present invention, the following provides definitions and explanations of relevant terms.

[0078] As used herein, when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length of the TIGIT protein, or the extracellular immunoglobulin variable region (IgV) domain, or a fragment containing the extracellular immunoglobulin variable region (IgV) domain, and further includes fusion proteins of TIGIT, such as fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG. However, it will be understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of the TIGIT protein can be naturally produced or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "TIGIT protein" or "TIGIT" is intended to include all such sequences, including the sequences shown and natural or artificial variants thereof. And, when describing a sequence fragment of the TIGIT protein, it not only includes the sequence fragment, but also includes the corresponding sequence fragment in its natural or artificial variant.

[0079] As used herein, the term EC 50 This refers to the concentration for 50% of maximal effect, which is the concentration that can produce 50% of the maximum effect.

[0080] As used herein, the term "antibody" refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined through a "J" region of about 12 or more amino acids, with the heavy chains further comprising a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 and C. H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)) interspersed with regions of relative conservedness called framework regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. H and V L) each form an antigen-binding site. The assignment of amino acids to each region or domain is defined according to Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917, Chothia et al., Nature 1989; 342: 878-883, or the IMGT numbering system, and refers to the definition in Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307. The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0081] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, capable of specifically binding to the same antigen bound by the full-length antibody and / or competing with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion." See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is incorporated herein by reference in its entirety and for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides having at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0082] As used herein, the term "Fd fragment" refers to a V H and C. H The term "Fv fragment" refers to an antibody fragment consisting of a single domain of an antibody. L and V H The term "dAb fragment" refers to an antibody fragment consisting of the V domain. H The term "Fab fragment" refers to an antibody fragment consisting of the V domain (Ward et al., Nature 341: 544-546 (1989)). L , V H , C L and C. H The term "F(ab')2 fragment" refers to an antibody fragment consisting of one domain, and the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region.

[0083] In some cases, the antigen-binding fragment of an antibody is L and V HSingle chain antibodies (e.g., scFvs) form monovalent molecules by forming pairs of linkers that allow the domains to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). Such scFv molecules include NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L It can have the general structure -COOH. A suitable prior art linker consists of a repeat sequence of the amino acid sequence GGGGS or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but also variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers of use in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0084] In some cases, the antigen-binding fragment of an antibody is a diantibody, i.e., V H and V LSee, for example, bivalent antibodies, in which the domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain to produce two antigen-binding sites (e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0085] In other cases, the antigen-binding fragment of an antibody is a "bispecific antibody," which refers to a complex formed by coupling arms of a first antibody (fragment) and a second antibody (fragment) or antibody-like, and the coupling methods include, but are not limited to, chemical reaction, gene fusion, and enzyme catalysis. The antigen-binding fragment of an antibody may be a "multispecific antibody," which includes, for example, a triabody, which is an antibody with three different antigen-binding specificities, and a tetrabody, which is an antibody with four different antigen-binding specificities. For example, a designed anchor protein overlapping protein (DARPin) is linked to an IgG antibody, scFv-Fc antibody fragment, or a combination thereof, such as shown in CN104341529A. An anti-IL-17a fynomer is linked to an anti-IL-6R antibody, such as shown in WO2015141862A1.

[0086] Those skilled in the art can obtain antigen-binding fragments of antibodies (e.g., the above-mentioned antibody fragments) from a given antibody (e.g., monoclonal antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 provided by the present invention) using known conventional techniques (e.g., recombinant DNA techniques or enzymatic or chemical cleavage) and specifically screen for antigen-binding fragments of antibodies in the same manner as used for whole antibodies.

[0087] As used herein, the terms "mono antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a series of highly homologous antibody molecules, i.e., a series of antibody molecules that are completely the same except for spontaneously occurring natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained by employing the hybridoma technique first published by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. nature, 1975; 256(5517): 495), but may also be obtained by employing recombinant DNA techniques (see, for example, US Patent 4,816,567).

[0088] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained after replacing all or part of the CDR regions of a human immunoglobulin (receptor antibody) with the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be a non-human antibody (e.g., mouse, rat or rabbit) of desired specificity, affinity or reactivity. Besides, some amino acid residues in the framework region (FR) of the receptor antibody can be replaced with the corresponding amino acid residues of a non-human antibody or replaced with amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody. For further details on humanized antibodies, see, for example, Jones et al., Nature 1986; 321: 522-525; Reichmann et al., Nature 1988; 332: 323-329; Presta, Curr. Op. Struct. Biol., 1992; 2: 593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes' [J]. Immunol. Today, 2000; 21(8): 397-402.

[0089] As used herein, the term "isolated" or "isolated" refers to being obtained by hand in a natural state. When an "isolated" substance or component occurs in nature, the natural environment in which it exists may have been altered, or the substance may have been isolated from its natural environment, or both. For example, a non-isolated polynucleotide or polypeptide may naturally occur in the body of an animal, and a highly purified version of the same polynucleotide or polypeptide isolated from such a natural state is referred to as isolated. The term "isolated" or "isolated" does not exclude mixtures of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0090] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, Cox plasmids, yeast artificial chromosomes (YACs), artificial chromosomes such as bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda bacteriophage or M13 bacteriophage, and animal viruses. Animal viruses as vectors include, but are not limited to, reverse transcribing retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papilloma baculoviruses (e.g., SV40). Vectors may contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes, and may further contain an origin of replication.

[0091] As used herein, the term "host cell" refers to cells useful for introducing a vector, including, but not limited to, prokaryotic cells, such as E. coli or Bacillus subtilis, fungal cells, such as yeast cells or Aspergillus, insect cells, such as S2 Drosophila cells or Sf9, or animal cells, such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.

[0092] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and an antigen against which it is directed. In one embodiment, an antibody that specifically binds to a particular antigen (or has specificity for a particular antigen) is one in which the antibody binds to the antigen at a concentration of about 10 -5 Less than M, e.g., about 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M or 10 -10 Affinity (K D ) to bind to the antigen.

[0093] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10 -5 Less than M, e.g., about 10 -6 Less than M, 10 -7 Less than 10 -8 Less than M, 10 -9 Less than M or 10 -10 The dissociation equilibrium constant (K D ) to bind to an antigen (e.g., TIGIT protein). D may be measured, for example, with a Fortebio molecular interaction instrument.

[0094] As used herein, the terms "monoclonal antibody" and "mono-antibody" have the same meaning and are used interchangeably, the terms "polyclonal antibody" and "poly-antibody" have the same meaning and are used interchangeably, and the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are usually represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0095] As used herein, the term "pharmacologically acceptable vector and / or excipient" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the subject active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents. For example, pH adjusting agents include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween®-80, and ionic strength enhancing agents include, but are not limited to, sodium chloride.

[0096] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a disease (e.g., tumor) preventive effective amount refers to an amount sufficient to prevent, inhibit, or delay the onset of a disease (e.g., tumor), and a disease treating effective amount refers to an amount sufficient to cure or at least partially inhibit a disease and its complications in a patient having the disease.

[0097] As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and reference to the terms "hybridoma" and "hybridoma cell line" further includes subclones and progeny of hybridomas.

[0098] The term "single drug dosage unit" refers to a single drug dosage form, e.g., an injection disposed in an ampoule, of the anti-TIGIT antibody and anti-PD-1-anti-VEGFA bispecific antibody described in the present invention, administered to a subject at a predetermined administration time (preferably on a per kg basis of the subject's body weight). In a specific embodiment of the present invention, the administration regimen includes, for example, administering the single drug dosage unit at a dosing frequency of twice a day to about once every two days, or once every three days, once every four days, once every five days, once every six days, once every ten days, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks.

[0099] In the present invention, unless otherwise specified, the above terms "first" (e.g., first protein functional domain, first linked fragment) and "second" (e.g., second protein functional domain, second linked fragment) are used merely for the sake of distinction in indication or clarity of expression, and do not have any meaning of indicating a typical order.

[0100] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that prevents the onset of a disease or promotes the resolution of a disease in a subject, as evidenced by a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of symptom-free periods of the disease, or a prevention of impairment or disability due to the affliction of the disease. A variety of methods known to those of skill in the art can be used to assess the ability of a therapeutic agent to promote resolution of a disease, for example, measuring the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0101] A "prophylactically effective amount" of a drug refers to any amount of drug that, when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-aggravated disease state) or at risk of cancer recurrence, inhibits the onset or recurrence of cancer. In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of cancer. "Inhibiting" the onset or recurrence of cancer refers to reducing the likelihood that cancer will develop or recur, or completely preventing the onset or recurrence of cancer. Effect of the Invention

[0102] The monoclonal antibody of the present invention can bind well and specifically to TIGIT with very strong affinity, reduce the inhibitory effect of TIGIT on immune cells, promote T cell activity, reverse NK cell depletion, and increase the killing effect of immune cells on tumors. It can be used to prepare drugs for inhibiting TIGIT and drugs for treating or preventing diseases such as tumors in combination with anti-PD-1-anti-VEGFA antibodies, and has good application prospects and market value. [Brief description of the drawings]

[0103] [Figure 1] 1 shows the results of detecting the binding activity of the 26B12H1L1, 26B12H2L2, 26B12H2L3, and 26B12H3L2 antibodies to TIGIT-mFc. [Diagram 2] 1 shows the results of detecting the binding activity of the 26B12H3L3, 26B12H1L4, 26B12H4L1, and 26B12H4L4 antibodies to TIGIT-mFc. [Diagram 3] 1 shows the results of activity detection of 26B12H1L1, 26B12H2L2, 26B12H2L3 and 26B12H3L2 antibodies competing with human CD155-hFc-biotin for binding to TIGIT-mFc. [Figure 4] 1 shows the results of activity detection of 26B12H3L3, 26B12H1L4, 26B12H4L1 and 26B12H4L4 antibodies competing with human CD155-hFc-biotin for binding to TIGIT-mFc. [Diagram 5]1 shows the results of determining the affinity constants of 26B12 H3L3 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 6] 1 shows the results of determining the affinity constants of 26B12 H1L1 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 7] 1 shows the results of determining the affinity constants of 26B12 H2L2 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 8] 1 shows the results of determining the affinity constants of 26B12 H2L3 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 9] 1 shows the results of determining the affinity constants of 26B12 H3L2 and TIGIT-mFc, where the antibody concentrations added in each pair of curves from top to bottom are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 10] 1 shows the results of determining the affinity constants of 26B12 H4L4 and TIGIT-mFc, with the antibody concentrations added being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively, for each pair of curves from top to bottom in the figure. [Figure 11] 1 shows the results of determining the affinity constants of 26B12 H1L4 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 12]1 shows the results of determining the affinity constants of 26B12 H4L1 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 13] 1 shows the results of determining the affinity constants of RG6058 and TIGIT-mFc, with the antibody concentrations added in each pair of curves from top to bottom being 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 14] Detection of the binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT by FACS. [Figure 15] This is detection by FACS of the activity of humanized antibodies 26B12H2L2 and RG6058 competing with CD155 for binding to TIGIT on the membrane surface of 293T-TIGIT cells. [Figure 16] This is detection by FACS of the activity of humanized antibodies 26B12H2L2 and RG6058 competing with CD112 for binding to TIGIT on the membrane surface of 293T-TIGIT cells. [Figure 17] Tumor model effect in hTIGIT-BALB / c recombinant mouse CT26. [Figure 18] Changes in body weight of the tumor model in hTIGIT-BALB / c recombinant mice CT26. [Figure 19] Effect of the combination of 26B12H2L2 and the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) on the tumor model in BALB / c-hPD1 / hTIGIT transgenic mice CT26. [Figure 20] Changes in tumor body weight in BALB / c-hPD1 / hTIGIT transgenic mice CT26 after combined administration of 26B12H2L2 and the anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM). [Figure 21]The combination of anti-TIGIT antibody and anti-PD-1-anti-VEGFA bifunctional antibody effectively neutralized the inhibition of the signaling pathway mediated by the binding of the corresponding target to its receptor.

[0104] Biological material for deposit: The hybridoma cell line LT019 (TIGIT-26B12) has been deposited in the China Center for Typical Culture Collection (CCTCC) since October 23, 2020, with the deposit number CTCCC NO: C2020208 and the deposit address is Wuhan University, Wuhan, China, with the postal code 430072. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. Those skilled in the art should understand that the following examples are merely for illustrating the present invention and do not limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, they will be performed according to the techniques or conditions described in the literature in the field (for example, see Sambrook J, et al., Molecular Cloning: A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory Press) or according to the product instructions. If the manufacturer is not specified, the reagents or equipment used may be commercially available general products. For example, 293T may be purchased from ATCC.

[0106] The BALB / c mice used in the following examples of the present invention are purchased from Guangdong Medical Experimental Animal Center.

[0107] In the following examples of the present invention, the positive control antibody RG6058 is used, and its sequence can be referred to as sequence 34 and sequence 36 in Chinese Patent Publication CN108290946.

[0108] In the following examples of the present invention, the combined anti-PD-1-anti-VEGFA bispecific antibody VP101 (hG1DM) used is produced by Zhongshan Kangfang Bio-Pharmaceutical Co., Ltd., and its sequence is derived from the antibody described in the published patent CN112830972A, of which, the full-length amino acid sequence of the heavy chain of VP101 (hG1DM) is shown in SEQ ID NO: 27, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 29. The VP101 (hG1DM) structure is an IgG-scFv, of which the IgG portion is an anti-VEGFA antibody and the scFv portion is an anti-PD-1 antibody; The HCDR1 sequence of the anti-VEGFA antibody is set forth in SEQ ID NO: 35, the HCDR2 sequence is set forth in SEQ ID NO: 36, the HCDR3 sequence is set forth in SEQ ID NO: 37, the VH sequence is set forth in SEQ ID NO: 31, the LCDR1 sequence of the anti-VEGFA antibody is set forth in SEQ ID NO: 38, the LCDR2 sequence is set forth in SEQ ID NO: 39, the LCDR3 sequence is set forth in SEQ ID NO: 40, and the VL sequence is set forth in SEQ ID NO: 33; Among them, the HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO: 45, the HCDR2 sequence is shown in SEQ ID NO: 46, the HCDR3 sequence is shown in SEQ ID NO: 47, the VH sequence is shown in SEQ ID NO: 41, the LCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO: 48, the LCDR2 sequence is shown in SEQ ID NO: 49, the LCDR3 sequence is shown in SEQ ID NO: 50, and the VL sequence is shown in SEQ ID NO: 43.

[0109] In the following examples of the present invention, the isotype control antibody used is human anti-hen egg lysozyme IgG (anti-HEL, i.e. human IgG, abbreviated as hIgG), and its variable region sequence is derived from Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-46). The hIgG1DM used in the examples is an isotype control antibody, i.e., anti-HEL having the hG1DM constant region sequence (SEQ ID NO: 55), and was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0110] In the following examples of the present invention, the CHO-aAPC-PDL1-PVR cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The CHO-aAPC-PDL1-PVR cell line was generated by viral infection of PD-L1 aAPC / CHO-K1 cells (purchased from Promega), and the virus was prepared using 3rd Generation Lentiviral Systems, see for example A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11): 8463-8471, in which the lentiviral expression vector used is pCDH-PVRFL-Puro (PVRFLGenebank ID: NP_006496.4, the vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbio, product number VT1480).

[0111] In the following examples of the present invention, the Jurkat-NFAT-PD1-TIGIT cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Jurkat-NFAT-PD1-TIGIT cell line was generated by viral infection of PD-1 effector cells (purchased from Promega), and the virus was prepared using 3rd Generation Lentiviral Systems, see for example A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11): 8463-8471, in which the lentiviral expression vector used is plenti6.3 / V5-TIGITFL-BSD (in which TIGIT, Genebank ID: NP_776160.2, and vector plenti6.3 / V5 TOPO, purchased from Invitrogen, product number K531520). EXAMPLES

[0112] Example 1: Preparation of anti-TIGIT antibody 26B12 1. Preparation of hybridoma cell line LT019 The antigen used to prepare anti-TIGIT antibodies is human TIGIT-mFc (TIGIT has Genbank ID: NP_776160.2, and the sequence of mFc is shown in SEQ ID NO: 51). Spleen cells from the immunized mice were taken and fused with mouse myeloma cells to produce hybridoma cells. Using human TIGIT-mFc as an antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies that specifically bind to TIGIT. Stable hybridoma cell lines were obtained by the limited dilution method from the hybridoma cells obtained by screening. The above hybridoma cell lines were named hybridoma cell lines LT019, and the monoclonal antibodies secreted by them were named 26B12.

[0113] The hybridoma cell line LT019 (also referred to as TIGIT-26B12) has been deposited at the China Center for Typical Culture Collection (CCTCC) since October 23, 2020, with the deposit number CTCCC NO: C2020208 and the deposit address is Wuhan University, Wuhan, China, with the postal code 430072.

[0114] 2. Preparation of anti-TIGIT antibody 26B12 The above LT019 cell line, which was prepared in CD medium (Chemical Defined Medium, containing 1% penicillin-streptomycin), was cultured under conditions of 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, centrifuged at high speed, filtered under vacuum using a microporous membrane, and purified with a HiTrap protein A HP column to obtain antibody 26B12.

[0115] Example 2: Sequence analysis of anti-TIGIT antibody 26B12 The mRNA was extracted from the LT019 cell line cultured in Example 1 by the method of cultured cell / bacteria total RNA extraction kit (Tiangen, product number DP430). cDNA was synthesized and PCR amplified according to the Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR kit instructions. TA cloning was performed directly on the PCR amplification product, and the specific procedure was performed by referring to the instructions for the pEASY-T1 Cloning Kit (Transgen CT101). The product of TA cloning was directly sequenced, and the sequencing results are as follows: The nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2, and the length of the fragment is 363 bp. The amino acid sequence it encodes is as shown in SEQ ID NO:1 and is 121 amino acids in length. Of these, the sequence of heavy chain HCDR1 is as shown in SEQ ID NO:3, the sequence of HCDR2 is as shown in SEQ ID NO:4, and the sequence of HCDR3 is as shown in SEQ ID NO:5. The nucleic acid sequence of the light chain variable region is as shown in SEQ ID NO:7 and is 321 bp in length. The amino acid sequence it encodes is as shown in SEQ ID NO:6 and is 107 amino acids in length. Among them, the sequence of the light chain LCDR1 is as shown in SEQ ID NO:8, the sequence of the LCDR2 is as shown in SEQ ID NO:9, and the sequence of the LCDR3 is as shown in SEQ ID NO:10.

[0116] Example 3: Design and preparation of the light and heavy chains of humanized anti-human TIGIT antibodies 1. Design of the light and heavy chains of the anti-human TIGIT humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4

[0117] Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained from Example 2, an antibody model was simulated in a computer, and then mutations were designed based on the model to obtain the variable region sequences of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 (the antibody constant region sequences were derived from the NCBI database, the heavy chain constant regions were all Ig gamma-1 chain C region, accession number: P01857, and the light chain constant regions were Ig kappa chain C region, accession number: P01834).

[0118] The designed variable region sequences are shown in Table A below. [Table 1]

[0119] (7) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 26B12H1L4 The above eight antibodies, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4, all have heavy chain variable region nucleic acid sequences that are 363 bp long and encoded amino acid sequences that are 121 aa long, and all have light chain variable region nucleic acid sequences that are 321 bp long and encoded amino acid sequences that are 107 aa long.

[0120] In addition, the above eight antibodies have the same HCDR1 to HCDR3 and LCDR1 to LCDR3, as follows: The sequence of HCDR1 is as shown in SEQ ID NO:3, the sequence of HCDR2 is as shown in SEQ ID NO:4, and the sequence of HCDR3 is as shown in SEQ ID NO:5. The sequence of LCDR1 is as set forth in SEQ ID NO:8, the sequence of LCDR2 is as set forth in SEQ ID NO:9, and the sequence of LCDR3 is as set forth in SEQ ID NO:10.

[0121] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 The heavy chain constant region used in both cases was the Ig gamma-1 chain C region, accession number: P01857, and the light chain constant region used in both cases was the Ig kappa chain C region, accession number: P01834.

[0122] The 26B12H1L1 heavy chain cDNA and light chain cDNA, 26B12H4L1 heavy chain cDNA and light chain cDNA, 26B12H2L2 heavy chain cDNA and light chain cDNA, 26B12H3L2 heavy chain cDNA and light chain cDNA, 26B12H2L3 heavy chain cDNA and light chain cDNA, 26B12H3L3 heavy chain cDNA and light chain cDNA, 26B12H1L4 heavy chain cDNA and light chain cDNA, 26B12H2L4 heavy chain cDNA and light chain cDNA, and 26B12H4L4 heavy chain cDNA and light chain cDNA were each cloned into pUC57simple (provided by GenScript) vector, and named pUC57simple-26B12H1, pUC57simple-26B12L1, pUC57simple, respectively. -26B12H4, pUC57simple-26B12L1, pUC57simple-26B12H2, pUC57simple-26B12L2, pUC57simple-26B12H3, pUC57simple-26B12L2, pUC57simple-26B12H2, pUC57simple-26B12L3, pUC57simple-26B12H3, pUC57simple-26B12L3, pUC57simple-26B12H1, pUC57simple-26B12L4, pUC57simple-26B12H2, pUC57simple-26B12L4, and pUC57simple-26B12H4, pUC57simple-26B12L4 were obtained. According to the standard techniques introduced in the "Molecular Cloning Laboratory Guide (2nd Edition)", the heavy and light chain full-length genes synthesized by EcoRI & HindIII enzyme digestion were subcloned into the expression vector pcDNA3.1 by enzyme digestion with restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-26B12H1, pcDNA3.1-26B12L1, pcDNA3.1-26B12H4, pcDNA3.1-126B12H2, pcDNA3.1-26B12L2, pcDNA3.1-26B12H3, pcDNA3.1-26B12L3 and pcDNA3.1-26B12L4, and the heavy and light chain genes of the recombinant expression plasmids were subjected to sequencing analysis.Next, the gene combinations (pcDNA3.1-26B12H1 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H5 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H6 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H8 / pcDNA3.1-26B12L4, pcDNA3.1-26B12H9 / pcDNA3.1-26B12L5, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L6, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L6, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L6, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L7, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L8, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L9, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L9, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L8, pcDNA3.1-26B12 pcDNA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4 and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4) were designed and co-transfected into 293F cells, respectively, and the culture medium was harvested and purified. After correct sequencing and verification, endotoxin-free expression plasmids were prepared and the plasmids were transiently co-transfected into HEK293 cells to express the antibodies. After 7 days of culture, the cell culture medium was harvested and affinity purified using a Protein A column to obtain humanized antibodies.

[0123] Example 4: Measurement of antibody binding activity to antigen TIGIT-mFc by ELISA method Experimental steps: Goat anti-mouse IgG Fc (purchased from Jackson, lot number: 132560), 2 μg / mL, was coated on the enzyme-labeled plate, and then incubated at 4°C for 16 hours. After incubation, the enzyme-labeled plate coated with goat anti-mouse IgG Fc was washed once with PBST, and then blocked for 2 hours with a PBST solution containing 1% BSA as a blocking solution for the enzyme-labeled plate. After blocking of the enzyme-labeled plate was completed, the plate was washed three times with PBST. Then, 1 μg / mL of human TIGIT-mFc, which is an antigen, was added, and the plate was left to incubate at 37°C for 30 minutes, and then the plate was washed three times with PBST. An antibody gradient-diluted with PBST solution was added to the wells of the enzyme-labeled plate, and the details of the antibody dilution gradient are shown in Tables 1 and 2. The enzyme-labeled plate to which the test antibody was added was incubated at 37°C for 30 minutes, and after the incubation was completed, the plate was washed three times with PBST. After washing the plate, HRP-labeled goat anti-human IgG Fc (purchased from Jackson, lot number: 128332) secondary antibody working solution diluted at a ratio of 1:5000 was added and incubated at 37°C for 30 minutes. After incubation was completed, the plate was washed four times with PBST, and TMB (Neogen, 308177) was added and color development was performed for 4 minutes in the dark, and the color reaction was terminated by adding a stop solution. The enzyme-labeled plate was immediately placed in a microplate reader, and the OD value of each well of the enzyme-labeled plate was read by selecting a light wavelength of 450 nm. Data analysis was performed using SoftMax Pro 6.2.1 software.

[0124] The results of antibody binding to the antigen TIGIT-mFc are shown in Figures 1 and 2. The OD values ​​for each dose are shown in Tables 1 and 2. Curve fitting was performed with the antibody concentration on the horizontal axis and the absorbance value on the vertical axis to determine the EC 50 The results are shown in Tables 1 and 2 and Figures 1 and 2. [Table 2] [Table 3]

[0125] The results show that antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 can all effectively bind to human TIGIT-mFc, the binding efficiency is dose-dependent, and the binding activity is equivalent to that of the same target positive drug RG6058, indicating that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 have the function of effectively binding to TIGIT.

[0126] Example 5: Determination of the activity of antibodies competing with CD155-hFc-biotin for binding to TIGIT-mFc by competitive ELISA method Experimental steps: 2 μg / mL TIGIT-mFc was coated onto the enzyme-labeled plate and incubated overnight at 4°C. After the incubation, the antigen-coated enzyme-labeled plate was washed once with PBST, and then blocked for 2 hours with 1% BSA in PBST as the enzyme-labeled plate blocking solution. After blocking of the enzyme-labeled plate was completed, the plate was washed three times with PBST. The antibody gradient diluted with PBST solution was added to the enzyme-labeled plate, and the antibody concentration details are shown in Tables 3 and 4. After incubation at room temperature for 10 minutes, an equal volume of 2 μg / mL (final concentration is 1 μg / mL) CD155-hFc-biotin (produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., lot number: 20170210, of which the GenBank number of CD155 is NP_006496.4, and the sequence of hFc is shown in SEQ ID NO: 54) was added, and after uniform mixing with the antibody, the enzyme-labeled plate was placed under 37 ° C conditions and incubated for 30 minutes. After incubation was completed, the plate was washed three times with PBST. After washing the plate, SA-HRP working solution diluted at a ratio of 1:4000 was added, and left to incubate for 30 minutes under 37 ° C conditions. After incubation, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added to develop the color in the dark for 5 min, and the color reaction was terminated by adding a stop solution. The enzyme-labeled plate was immediately placed in a microplate reader, and the OD value of each well of the enzyme-labeled plate was read by selecting a light wavelength of 450 nm. Data analysis was performed using SoftMax Pro 6.2.1 software.

[0127] The activity results of the antibodies competing with CD155-hFc-biotin for binding to TIGIT-mFc are shown in Tables 3 and 4. The EC of the antibodies competing with CD155-hFc-biotin for binding to TIGIT-mFc was calculated by curve fitting with the antibody concentration on the horizontal axis and the absorbance value on the vertical axis. 50 The results are shown in Tables 3 and 4 below and in Figures 3 and 4.

[0128] [Table 4] [Table 5]

[0129] The results show that under the same experimental conditions, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 can respectively compete with CD155-hFc-biotin for binding to antigen TIGIT-mFc, and the activity is comparable to that of the same target positive drug RG6058, suggesting that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 have the function of competing with CD155-hFc-biotin for effective binding to TIGIT-mFc.

[0130] Example 6: Measurement of kinetic parameters of binding of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1 and RG6058 to antigen TIGIT-mFc using a Fortebio molecular interaction instrument The sample dilution buffer is PBS, 0.02% Tween (registered trademark)-20, 0.1% BSA, pH 7.4. TIGIT-mFc is immobilized on the AMC sensor at a concentration of 3 μg / mL, the time is 50 s, the sensor is equilibrated in the buffer for 60 s, the TIGIT-mFc immobilized on the sensor binds to the antibody, the concentration is 0.06-5 nM (3-fold dilution), the time is 120 s, the protein dissociates in the buffer, the time is 300 s. The sensor employs 10 mM glycine and is regenerated with a solution of pH = 1.7. The detection temperature is 37 degrees, the detection frequency is 0.3 Hz, and the sample plate vibration speed is 1000 rpm. The data was fitted and analyzed with a 1:1 model to obtain the affinity constant.

[0131] The results of measuring the affinity constant of the humanized antibody (as a control antibody) to TIGIT are shown in Table 5, and the detection results are shown in Figures 5 to 13. [Table 6]

[0132] The results show that the affinity constants of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1 and RG6058 to TIGIT-mFc are 9.64E-11 M, 1.64E-11 M, 8.40E-12 M, 4.85E-11 M, 5.40E-11 M, 3.69E-11 M, 4.63E-11 M, 8.57E-12 M and 3.16E-11 M, respectively.

[0133] The results show that the affinity of each TIGIT antibody to bind to TIGIT-mFc is, in order from strongest to weakest, 26B12H2L2, 26B12H4L1, 26B12H1L1, RG6058, 26B12H4L4, 26B12H1L4, 26B12H2L3, 26B12H3L2, 26B12H3L3. Among them, the affinity of humanized antibody 26B12H2L2, 26B12H4L1, 26B12H1L1 is stronger than that of positive drug RG6058, while the affinity of 26B12H4L4 is equivalent to that of positive drug RG6058.

[0134] Example 7: Detection of binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT by FACS Testing Method: The TIGIT vector was plenti6.3-TIGITFL-BSD (TIGIT is Genbank ID: NP_776160.2), and we asked GenScript Gene to synthesize the full-length human TIGIT cDNA sequence, named it TIGITFL, and cloned it into the pUC57simple (provided by GenScript) vector to obtain the pUC57simple-TIGITFL plasmid. Using the pUC57simple-TIGITFL plasmid synthesized by double enzyme digestion with BamHI & XhoI, the TIGITFL target gene fragment was recovered and subcloned into the plenti6.3 expression vector (vector pLenti6.3 was purchased from Invitrogen) by the restriction site BamHI & XhoI, and transfected into 293T cells. We obtained the cell line 293T-TIGIT cells that stably express TIGIT by screening.

[0135] 293T-TIGIT cells (DMEM + 10% FBS) were harvested, centrifuged for 5 min, the supernatant was removed, resuspended, and counted for number and viability (P7, 95.79%). The cells were diluted and added to a clear pointed bottom 96-well plate with 30 w of cells per well, 200 μL of 1% PBSA per tube, centrifuged for 5 min, and the supernatant was removed. Depending on the experimental design, 100 μL of antibodies were added per well (final concentrations of 300 nM, 100 nM, 33.3 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM), and blank and isotype controls were designed and incubated on ice for 60 min. 200 μL of 1% PBSA per tube was added, centrifuged for 5 min, the supernatant was removed, and the cells were washed twice. FITC goat anti-human IgG antibody (purchased from Jackson, product number: 109-095-098, diluted 500-fold with PBSA) was added to each sample, incubated on ice in the dark for 40 min, 200 μL of PBSA was added to each tube, centrifuged for 5 min, and the supernatant was removed. The cells were resuspended by adding 200 μL of PBSA, transferred to a flow tube, and the average fluorescence intensity of the cells at each concentration was detected by a flow cytometer. [Table 7]

[0136] The experimental results are shown in Table 6 and FIG. 14. The EC 50 The EC of the humanized antibody 26B12H2L2, which binds to the cell membrane surface antigen TIGIT, is 1.257 nM. 50 is 0.917 nM. The experimental results showed that the binding ability of the humanized antibody 26B12H2L2 to the cell membrane surface antigen TIGIT was stronger than that of the positive control antibody RG6058.

[0137] Example 8: Detection of the competitive activity of humanized antibodies 26B12H2L2 and RG6058 with CD155 or CD112 for binding to the 293T-TIGIT cell membrane surface antigen TIGIT by FACS Experimental method: 293T-TIGIT cells were harvested, centrifuged for 5 min, the supernatant was removed, resuspended, and the number and viability were counted (94.95%). The cells were diluted and added to a clear pointed bottom 96-well plate at 300,000 cells per well, 200 μL of 1% PBSA was added per tube, centrifuged for 5 min, and the supernatant was removed. According to the experimental design, 100 μL of antibody (final concentration 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM) was added per well accordingly, and blank control and isotype control were designed and incubated on ice for 30 min. CD155 (final concentration 10 nM, produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., lot number: 20190726, of which the GenBank number of CD155 is NP_006496.4) or CD112 (final concentration 30 nM, produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., lot number: 20190726, of which the GenBank number of CD112 is NP_001036189.1) was added to each sample and incubated on ice in the dark for 60 min. 200 μL of 1% PBSA was then added to each tube, centrifuged for 5 min, the supernatant was removed, and the tube was washed twice. APC goat anti-mouse IgG (purchased from Biolegend, lot number 405308, minimal x-reactivity) antibody (diluted 300-fold with PBSA) was added to each sample, incubated on ice in the dark for 40 min, 200 μL of PBSA was added to each tube, centrifuged for 5 min, and the supernatant was removed. The cells were resuspended by adding 200 μL of PBSA, transferred to a flow tube, and the average fluorescence intensity of the cells at each concentration was detected by a flow cytometer. The experimental results are shown in Table 7 and Figure 15, and Table 8 and Figure 16, respectively. [Table 8] [Table 9]

[0138] Results show that the EC 50 The EC of humanized antibody 26B12H2L2, which competes with CD155 for binding to TIGIT, is 1.212 nM. 50 The EC of the positive control antibody RG6058, which competes with CD112 for binding to TIGIT, was 1.049 nM. 50 The EC of humanized antibody 26B12H2L2, which competes with CD112 for binding to TIGIT, is 1.224 nM. 50 is found to be 1.140 nM. The results show that the ability of humanized antibody 26B12H2L2 to compete with CD155 or CD112 for binding to the cell membrane surface antigen TIGIT is stronger than that of the positive control antibody RG6058.

[0139] Example 9: Therapeutic effect of 26B12H2L2 inoculated with CT26 mouse-transplanted tumors in hTigit-BALB / c transgenic mice The hTigit-BALB / c transgenic mice (mice were purchased from Jiangsu Jixi Yaokang Biotechnology Co., Ltd., and the normal mouse TIGIT gene of the purchased transgenic mice was replaced with the human TIGIT gene) were inoculated with 500,000 CT26 cells (mouse colon cancer 0 cell line, purchased from ATCC) on the back. The specific step of the experiment is to establish a mouse tumor model by inoculating the inoculated mice with 25 million CT26 cells / mL at 200 μL per mouse. The experimental mice were divided into an isotype control group (dosage 20 mg / kg, administration method intraperitoneal injection (ip), twice a week) and an experimental group (dosage 20 mg / kg, administration method intraperitoneal injection (ip), twice a week), with 8 mice per group. The specific schedule is shown in Table 9. [Table 10]

[0140] The experimental results are shown in FIG. The results show that 26B12H2L2 significantly reduced tumor volume in the hTIGIT-BALB / c transgenic mouse CT26 tumor model. The results indicate that 26B12H2L2 has strong therapeutic efficacy in hTIGIT-BALB / c transgenic mouse CT26 tumor model and may be used for the treatment and / or prevention of tumors, especially colon cancer. At the same time, as shown in FIG. 18, 26B12H2L2 had no effect on the body weight of hTIGIT-BALB / c transgenic mice as a CT26 tumor model, indicating that the 26B12H2L2 antibody has no toxicity or side effects on mice.

[0141] Example 10: Effective tumor treatment with anti-TIGIT antibody in combination with anti-PD-1-anti-VEGFA bifunctional antibody To detect the in vivo antitumor activity of anti-TIGIT antibody in combination with anti-PD-1-anti-VEGFA bifunctional antibody VP101 (hG1DM), CT26 cells (human colon cancer cells, purchased from Jiangsu Jiaxing Pharmaceuticals Co., Ltd.) were first subcutaneously inoculated into 5- to 7-week-old female BALB / c-hPD1 / hTIGIT mice (purchased from Jiangsu Jiaxing Pharmaceuticals Co., Ltd.) to produce tumors with an average tumor volume of 80 mm. 3 ~120 mm 3 When the tumor volume reached 100 mg / kg, the mice were randomly divided into 4 groups, with 6 mice per group. The day of grouping was designated as D0, and administration began on the day of grouping, D0. The administration method for the combined administration group was to prepare the drugs separately and administer them before and after (there is no specific requirement for the administration order and interval time, and one drug is administered after another drug). The modeling and specific administration method are shown in Table 10. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated. [Table 11]

[0142] The results are shown in Figure 19. As can be seen from the results, compared with the isotype control antibody hIgG, both VP101(hG1DM) and 26B12H2L2 could effectively inhibit the growth of mouse tumors, and the VP101(hG1DM)+26B12H2L2 group showed a combined antitumor effect in the model, with the combined tumor inhibition being superior to that of the test drug alone group. *p<0.05, **p<0.01, ***p<0.0001, Two-way ANOVA (Bnoferroni posttest) Furthermore, as shown in FIG. 20, the tumor-bearing mice tolerated the test drugs VP101 (hG1DM) and 26B12H2L2 well, either alone or in combination, and there was no effect on the body weight of the tumor-bearing mice in each group.

[0143] Example 11: Combination of anti-TIGIT antibody and anti-PD-1-anti-VEGFA bifunctional antibody effectively neutralizes the inhibition of signal transduction pathways mediated by the binding of the corresponding targets to their receptors CHO-aAPC-PDL1-PVR cells (constructed by Yasuho Bio) were collected and centrifuged at 170 x g for 5 min. The supernatant was discarded, and the cells were resuspended in complete medium (Ham's F-12 + 10% FBS). The cell number and viability were counted, and the CHO-aAPC-PDL1-PVR cells were cultured at 4 × 10 4 Add 100 μL / well to a 96-well black plate, add 150 μL of 1× PBS to the edge wells, and incubate overnight at 37℃ in an incubator. Collect Jurkat-NFAT-PD1-TIGIT cells (constructed by Yasuho Bio) and centrifuge at 110×g for 5 min to discard the supernatant. Then resuspend the cells in the medium (RPMI 1640+10%FBS) to analyze the cell number and viability. Discard the liquid in the 96-well black plate, add 30 μL / well of Jurkat-NFAT-PD1-TIGIT cells at 5×10 4 A549 lung cancer cells (purchased from the Cell Resource Center of Shanghai Institutes of Life Sciences, Chinese Academy of Sciences, product number: SCSP-503) were collected and centrifuged at 170 x g for 5 min. The supernatant was discarded, and the medium was analyzed to resuspend the cells, count the cell number and viability, and 1 × 10 A549 cells were added in 30 μL / well. 410 μL / well was added, and the antibody was diluted according to the experimental design (the final concentrations of VP101 (hG1DM) and 26B12H2L2 in the single drug group were 3 nM, 30 nM, 300 nM, and 1000 nM, respectively, and the final concentrations of VP101 (hG1DM) and 26B12H2L2 in the combination group VP101 (hG1DM) + 26B12H2L2 were 3 nM, 30 nM, 150 nM, 300, and 1000 nM, respectively). The antibody was added at 20 μL / well, and an isotype control group and a negative control group were set up. The final volume was 80 μL / well, and the cells were cultured in an incubator at 37°C for 6 h. After 6 h, 80 μL of Firefly Glo Luciferase Reporter Gene Assay Kit (purchased from Yeasen, product number: 11404ES80) reaction solution was added, and the cells were incubated for 5 min to 6 h. After a min incubation, the culture plate was placed in the instrument to measure the relative light units (RLU). The results are shown in FIG.

[0144] As can be seen from the results, compared with the isotype control antibody hIgG1DM, VP101(hG1DM) alone, 26B12H2L2 alone and the combination of VP101(hG1DM)+26B12H2L2 can effectively neutralize the inhibitory effect on the signal transduction pathway mediated by the binding of the corresponding target with its receptor, and enhance the expression of luciferase. Among them, the RLU values ​​of the combination groups of 150 nM and 300 nM were significantly higher (>3 times) than those of the single-agent groups of 300 nM and 1000 nM, indicating that the neutralizing activity of the combination of VP101(hG1DM)+26B12H2L2 was significantly superior to that of the single-agent groups.

[0145] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that, in accordance with all teachings disclosed, modifications and substitutions to these details are possible, and these modifications are within the scope of the claims of the present invention. The full scope of the present invention is defined by the appended claims and any equivalents thereof.

[0146] SEQUENCE LISTING (Note: CDR sequences are underlined) Amino acid sequence of 26B12VH EVQLQESGPGLVKPSQSLSLTCTVT GHSFTSDYA WNWIRQFPGNRLEWMGY ISYSDST NYNPSLKSRISITRDTSKNQFFLQMNSVTTEDTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 1) Nucleotide sequence of 26B12VH GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGAAACCCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACT GGCCACTCATTCACCAGTGATTATGCC TGGAACTGGATCCGGCAGTTTCCAGGAAACAGACTGGAGTGGATGGGCTAC ATAAGCTACAGTGATAGCACT AACTACAACCCATCTCTCAAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCTTGCAGATGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGT GCAAGATTGGACTATGGTAACTACGGTGGGGCTATGGACTAC TGGGGTCAAGGGACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 2) HCDR1 of 26B12VH: GHSFTSDYA (SEQ ID NO: 3) HCDR2 of 26B12VH: ISYSDST (SEQ ID NO: 4) HCDR3 of 26B12VH: ARLDYGNYGGAMDY (SEQ ID NO:5) Amino acid sequence of 26B12VL DIVLTQSHEFMSTSLRDRVSITCKSS QHVSTA VAWYQQKPGQSPKLLIY SAS YRYTGVPDRFTGSGSGTDFTFTISSVKAEDLAVYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO:6) Nucleotide sequence of 26B12VL GATATTGTGCTAACTCAGTCTCACGAATTCATGTCCACCTCATTACGAGACAGGGTCAGCATCACCTGCAAATCCAGT CAACATGTGAGTACTGCT GTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTAC TCGGCATCC TACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCATGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGT CAGCAACATTATATTACTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATAAAA (SEQ ID NO: 7) LCDR1 of 26B12VL: QHVSTA (SEQ ID NO: 8) LCDR2 of 26B12VL: SAS (SEQ ID NO: 9) LCDR3 of 26B12VL: QQHYITPWT (SEQ ID NO: 10) Amino acid sequence of 26B12H1 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WNWIRQFPGKGLEWIGY ISYSDST NYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 11) Nucleotide sequence of 26B12H1 GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCT GGCCACAGCTTCACATCCGACTACGCC TGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATCGGCTAC ATCTCTTATAGCGACTCCACC AACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 12) Amino acid sequence of 26B12H2 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WSWIRQPPGKGLEWIGY ISYSDST NYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 13) Nucleotide sequence of 26B12H2 GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCC GGCCACTCTTTTACAAGCGACTACGCC TGGTCTTGGATCAGGCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTAC ATCTCCTATTCTGACAGCACC AACTATAATCCCTCCCTGAAGTCTCGGGTGACCATCTCTAGAGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 14) Amino acid sequence of 26B12H3 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WSWIRQPPGKGLEWIGY ISYSDST NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 15) Nucleotide sequence of 26B12H3 GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCC GGCCACTCTTTTACAAGCGACTACGCC TGGTCTTGGATCAGACAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTAC ATCTCCTATTCTGACAGCACC AACTATAATCCCTCCCTGAAGTCTAGAGTGACCATCTCTGTGGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 16) Amino acid sequence of 26B12H4 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WNWIRQFPGKGLEWMGY ISYSDST NYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 17) Nucleotide sequence of 26B12H4 GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCT GGCCACAGCTTCACATCCGACTACGCC TGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATGGGCTAC ATCTCTTATAGCGACTCCACC AACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 18) Amino acid sequence of 26B12L1 DIQMTQSPKSLSTSVGDRVTITCRSS QHVSTA VAWYQQKPGKSPKLLIY SAS YRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 19) Nucleotide sequence of 26B12L1 GACATCCAGATGACCCAGTCCCCTAAGTCCCTGTCTACAAGCGTGGGCGATCGGGTGACCATCACATGTAGAAGCTCC CAGCACGTGTCTACCGCA GTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAT TCCGCCTCT TACAGGTATTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACC TTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 20) Amino acid sequence of 26B12L2 DIQMTQSPSSLSASVGDRVTITCRSS QHVSTA LAWYQQKPGKSPKLLIY SAS SRYSGVPDRFSGSGSGTDFTFTISSLQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO:21) Nucleotide sequence of 26B12L2 GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTAGATCTAGC CAGCACGTGTCTACAGCC CTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAC TCCGCCTCC TCTAGGTATTCTGGAGTGCCAGACCGGTTTTCCGGCTCTGGCAGCGGCACCGATTTCACCTTTACAATCAGCTCCCTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTATATCACCCCATGGACC TTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 22) Amino acid sequence of 26B12L3 DIQMTQSPSSLSASVGDRVTITCRAS QHVSTA LAWYQQKPGKAPKLLIY SAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO:23) Nucleotide sequence of 26B12L3 GACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGATAGGGTGACCATCACATGTAGAGCCTCT CAGCACGTGAGCACAGCC CTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTAT AGCGCCTCT AGCCTGCAGTCCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGAACCGACTTTACCCTGACAATCTCCTCTCTGCAGCCAGAGGATTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACC TTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 24) Amino acid sequence of 26B12L4 DIQMTQSPKSMSTSVGDRVTITCRSS QHVSTA VAWYQQKPGKSPKLLIY SAS YRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 25) Nucleotide sequence of 26B12L4 GACATCCAGATGACCCAGTCCCCTAAGTCCATGTCTACAAGCGTGGGCGACAGGGTGACCATCACATGTAGAAGCTCC CAGCACGTGTCTACCGCA GTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAT TCCGCCTCT TACAGGTATTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACC TTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 26) Amino acid sequence of the heavy chain of VP101(hG1DM) (SEQ ID NO:27) Nucleic acid sequence of the heavy chain of VP101(hG1DM) Amino acid sequence of the light chain of VP101(hG1DM) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29) Nucleotide sequence of the light chain of VP101(hG1DM) (SEQ ID NO:30) Amino acid sequence of bevacizumab heavy chain variable region (bevacizumab-Hv): (123 aa) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 31) Nucleotide sequence of the bevacizumab heavy chain variable region: (369 bp) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTCTCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAATGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGGATCAACACTTACACCGGGGAACCTACCTATGCAGCCGA CTTTAAGCGGCGGTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGAACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCCCACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGACTCTGGTGACAGTGAGCAGC (SEQ ID NO: 32) Amino acid sequence of bevacizumab light chain variable region (bevacizumab-Lv): (107 aa) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 33) Nucleotide sequence of the bevacizumab light chain variable region: (321 bp) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGGGCGACAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTCACTAGCTCCCTGCA CTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATCTGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCGCTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAGGGGACTAAAGTCGAGATCAAG (SEQ ID NO: 34) ベバシズマブ HCDR1 of the heavy chain variable region: GYTFTNYG (SEQ ID NO: 35) ベバシズマブ HCDR2 of the heavy chain variable region: INTYTGEP (SEQ ID NO: 36) ベバシズマブ HCDR3 of the heavy chain variable region: AKYPHYYGSSHWYFDV (SEQ ID NO: 37) ベバシズマブ LCDR1 of the light chain variable region: QDISNY (SEQ ID NO: 38) ベバシズマブ LCDR2 of the light chain variable region: FTS (SEQ ID NO: 39) ベバシズマブ LCDR3 of the light chain variable region: QQYSTVPWT (SEQ ID NO: 40) Amino acid sequence of the heavy chain variable region of humanized monoclonal antibody 14C12H1L1: (118 aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 41) Nucleotide sequence of the heavy chain variable region of humanized monoclonal antibody 14C12H1L1: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 42) Amino acid sequence of 14C12H1L1(M) light chain variable region: (108 aa, mutation sites in the amino acid sequence based on 14C12H1L1 are underlined) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK R (SEQ ID NO:43) Nucleotide sequence of the 14C12H1L1(M) light chain variable region: GATATCCAGATGACCCAGTCCCCCTCCTCTATGTCTGCCAGCGTGGGCGACCGGGTGACCTTCACATGTAGAGCCTCCCAGGATATCAACACCTACCTGTCTTGGTTTCAGCAGAAGCCCGCAAGAGCCCTAAGACACTGATCTATCGGGCCAATAGACTGGTGA GCGGAGTGCCTTCCCGGTTCTCCGGCTCTGGCAGCGGACAGGACTATACCCTGACAATCAGCTCCCTGCAGCCAGGGATATGGCCACATACTATTGCCTGCAGTATGACGAGTTCCCCCTGACCTTCGGGGCTGGCACTAAGCTGGAGCTGAAAAGA (SEQ ID NO: 44) HCDR1 of the heavy chain variable region of 14C12H1L1(M): GFAFSSYD (SEQ ID NO: 45) HCDR2 of the heavy chain variable region of 14C12H1L1(M): ISGGGRYT (SEQ ID NO:46) HCDR3 of the heavy chain variable region of 14C12H1L1 (M): ANRYGEAWFAY (SEQ ID NO: 47) LCDR1 of the 14C12H1L1 (M) light chain variable region: QDINTY (SEQ ID NO: 48) LCDR2 of the 14C12H1L1 (M) light chain variable region: RAN (SEQ ID NO: 49) LCDR3 of the 14C12H1L1 (M) light chain variable region: LQYDEFPLT (SEQ ID NO: 50) Amino acid sequence of mFc PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERRTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 51) Amino acid sequence of the first linked fragment GGGGSGGGGSGGGGS (SEQ ID NO:52) Amino acid sequence of the second ligated fragment GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:53) Amino acid sequence of hFc THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54) Amino acid sequence of the heavy chain constant region of hIgG1DM ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55)

Claims

1. A combination pharmaceutical for treating a tumor, comprising: an effective amount of an anti-TIGIT antibody and an effective amount of an anti-PD-1-anti-VEGFA bispecific antibody; The anti-TIGIT antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 3 to 5, respectively, and a light chain variable region comprising LCDR1 to LCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 8 to 10, respectively; The anti-PD-1 / anti-VEGFA bispecific antibody a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; wherein the first protein functional domain is an immunoglobulin and the second protein functional domain is a single-chain antibody; or the first protein functional domain is a single-chain antibody and the second protein functional domain is an immunoglobulin; the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 35 to 37, respectively, and the light chain variable region amino acid sequences of the immunoglobulin comprise LCDR1 to LCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 38 to 40, respectively; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 45 to 47, respectively, and the light chain variable region of the single-chain antibody comprises LCDR1 to LCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 48 to 50, respectively; Or, the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 45 to 47, respectively, and the light chain variable region comprises LCDR1 to LCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 48 to 50, respectively; The single-chain antibody comprises HCDR1 to HCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 35 to 37, respectively, and its light chain variable region comprises LCDR1 to LCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 38 to 40, respectively; The immunoglobulin is a human IgG1 subtype. Concomitant medications.

2. The immunoglobulin in the anti-PD-1 / anti-VEGFA bispecific antibody comprises a heavy chain constant region, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin comprises a combination of the following mutations: L234A and L235A, or L234A and G237A, or L235A and G237A, or The pharmaceutical combination of claim 1, having one of L234A, L235A, and G237A.

3. The combination drug of claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO:

25.

4. The heavy chain variable region and the light chain variable region of the anti-TIGIT antibody are selected from the group consisting of the following 1) to 9): 1) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 6; 2) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 19; 3) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 19; 4) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 21; 5) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 23; 6) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 21; 7) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 23; 8) the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 25; or 9) The amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:

25. The combined pharmaceutical composition according to any one of claims 1 to 3, wherein the variable region is selected from a heavy chain variable region and a light chain variable region.

5. A combination pharmaceutical described in any one of claims 1 to 3, wherein the anti-TIGIT antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being an Ig gamma-1 chain C region, and the light chain constant region being an Ig kappa chain C region.

6. the anti-PD-1 / anti-VEGFA bispecific antibody amino acid sequence: The heavy chain variable region of the immunoglobulin shown in SEQ ID NO: 31, the light chain variable region of the immunoglobulin whose amino acid sequence is shown in SEQ ID NO: 33, the heavy chain variable region of the single chain antibody shown in SEQ ID NO: 41, and the light chain variable region of the single chain antibody shown in SEQ ID NO: 43 Or, The heavy chain variable region of the immunoglobulin shown in SEQ ID NO: 41, the light chain variable region of the immunoglobulin whose amino acid sequence is shown in SEQ ID NO: 43, the heavy chain variable region of the single chain antibody shown in SEQ ID NO: 31, and the light chain variable region of the single chain antibody shown in SEQ ID NO: 33 The combined pharmaceutical composition according to any one of claims 1 to 3, comprising:

7. The anti-PD-1 / anti-VEGFA bispecific antibody a first protein functional region that targets PD-1; a second protein functional region that targets VEGFA; wherein the first protein functional domain is a single chain antibody and the second protein functional domain is an immunoglobulin; The immunoglobulin comprises a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 31, and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO: 33; the single-chain antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 41 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 43; The single chain antibody is linked to the C-terminus or N-terminus of an immunoglobulin heavy chain, the first protein functional region is linked to the second protein functional region via a first linking fragment, and the heavy chain variable region of the single chain antibody is linked to the light chain variable region of the single chain antibody via a second linking fragment, and the first linking fragment and the second linking fragment are homologous or different; The combined medicine according to any one of claims 1 to 3.

8. The combination pharmaceutical described in claim 7, wherein the amino acid sequences of the first linked fragment and the second linked fragment are independently selected from SEQ ID NO: 52 and SEQ ID NO:

53.

9. The combination drug described in claim 7, wherein the amino acid sequences of the first linked fragment and the second linked fragment are shown in sequence number 53.

10. The anti-PD-1 / anti-VEGFA bispecific antibody, a first protein functional region linked directly or via a linking fragment to a second protein functional region, and / or a heavy chain variable region of the single chain antibody linked directly or via a linking fragment to a light chain variable region of the single chain antibody; The combined pharmaceutical composition according to any one of claims 1 to 3, comprising:

11. The combination pharmaceutical of claim 10, wherein the anti-PD-1 / anti-VEGFA bispecific antibody comprises a linked fragment that is (GGGGS)n (wherein n is 1, 2, 3, 4, 5, or 6).

12. The anti-PD-1 / anti-VEGFA bispecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:27 and a light chain having the amino acid sequence set forth in SEQ ID NO:

29. The combined pharmaceutical composition of claim 1.

13. The combination pharmaceutical of claim 1, further comprising an effective amount of an antitumor chemotherapeutic agent.

14. The combination pharmaceutical of claim 13, wherein the antitumor chemotherapeutic agent comprises an alkylating agent, an anthracycline agent, an antihormonal agent, an aromatase inhibitor, an antiandrogen agent, a protein kinase inhibitor, a lipid kinase inhibitor, an antisense oligonucleotide, a ribozyme, an antimetabolite, a topoisomerase inhibitor, a cytotoxic agent or an antitumor antibiotic, a proteasome inhibitor, an antimicrotubule agent, an EGFR antagonist, a retinoid, a tyrosine kinase inhibitor, a histone deacetylase inhibitor, and combinations thereof.

15. The pharmaceutical combination of any one of claims 1 to 3, wherein the therapeutic combination further comprises one or more pharmaceutically acceptable excipients.

16. The combination drug according to any one of claims 1 to 3, wherein the tumor is selected from one or more of pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, plasma cell carcinoma, head and neck cancer, brain tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, lung cancer, breast cancer, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, malignant melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, intestinal cancer, hepatocellular carcinoma, renal cancer, peritoneal cancer, leukemia, and osteosarcoma.

17. The method of claim 16, wherein the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer. b) the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; c) the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma, or d) the tumor is a solid tumor of MSI-H / dMMR phenotype selected from one or more of colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumor; The combined pharmaceutical composition of claim 16.

18. The combination drug of claim 16, wherein the tumor is a malignant tumor.

19. The combination drug described in claim 16, wherein the tumor is hepatocellular carcinoma.

20. The combination drug described in any one of claims 1 to 3, wherein the effective amount of the anti-TIGIT antibody is 1 mg to 1,000 mg.

21. The combination pharmaceutical of claim 1, wherein the effective amount of the anti-PD-1 / anti-VEGFA bispecific antibody is 1 mg to 1,000 mg.

22. The combination drug of claim 1, wherein the effective amount of the anti-TIGIT antibody is 4 mg / kg.

23. The combination pharmaceutical of claim 1, wherein the effective amount of the anti-PD-1 / anti-VEGFA bispecific antibody comprises 1 mg / kg.

24. An effective amount of an anti-TIGIT antibody and an anti-PD-1 / anti-VEGFA bispecific antibody is administered twice a day to about once every two days, or once every three days, once every four days, once every five days, once every six days, once every ten days, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks, The administration method is intravenous infusion or intravenous injection. The combined medicine according to any one of claims 1 to 3.

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