Drug Compositions and Uses

Abstract

The pharmaceutical composition comprises an anti-TIGIT antibody or an antigen-binding fragment thereof, and an anti-CTLA4-anti-PD-1 bispecific antibody or an antigen-binding fragment thereof, in which the antibody heavy chain variable region comprises HCDR1 to HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 3 to 5, respectively, and the antibody light chain variable region comprises LCDR1 to LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8 to 10, respectively.

Description

[Technical field]

[0001] The present invention belongs to the pharmaceutical field and relates to a pharmaceutical composition comprising an anti-TIGIT antibody or an antigen-binding fragment thereof, and an anti-CTLA4-anti-PD-1 bispecific antibody or an antigen-binding fragment thereof. [Background technology]

[0002] TIGIT (also known as T cell Ig and ITIM domain, WUCAM, Vstm3, and VSIG9) is a member of the poliovirus receptor (PVR) / Nectin family. TIGIT consists of an extracellular immunoglobulin variable domain (IgV), a type I transmembrane domain, and an intracellular domain with classical immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoglobulin tyrosine-based tail (ITT) motifs. TIGIT is highly expressed on lymphocytes, particularly on 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 known as PVR, Necl5 or Tage4), CD112 (also known as PVRL2 / nectin2) and CD113 (also known as PVRL3) are TIGIT-binding ligands (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors[J]. Nature Reviews Immunology, 2015, 15(4): 243-254), where CD155 is a high-affinity ligand for TIGIT. In NK cells, TIGIT-binding ligands CD155 and CD112 can inhibit the killing effect 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). On the other hand, simultaneous inhibition of both PD-1 and TIGIT has been reported to enhance the killing effect 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 studies have demonstrated that TIGIT acts as an immune checkpoint for NK cells, and that the inhibitory receptor TIGIT leads to NK cell exhaustion during tumor progression, and that anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and be used for immunotherapy of many 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] It has also been reported that the addition of CD96 blockade to TIGIT blockade alone or in combination with PD-1 blockade significantly suppressed 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 blockade and / or PD-1 blockade increases the ability of TILs to produce cytokines in ovarian, endometrioma, 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 Res 2019;7:257-68).

[0005] Anti-TIGIT antibody drugs are expected to have a wide range of applications as novel immune checkpoint antibody drugs and can be used in tumor immunotherapy. Tiragolumab, developed by Roche, is in Phase 3 clinical trials, and in a Phase 2 clinical study treating patients with PD-L1-positive metastatic non-small cell lung cancer (NSCLC), the combination of Tiragolumab and Tecentriq was well tolerated, the risk of disease progression was reduced by 43%, and the combination effect was remarkable (Exit C. Roche to present first clinical data on novel anti-TIGIT cancer immunotherapy tiragolumab at ASCO[J]). Existing clinical information has demonstrated that 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-lymphoma, and plasma cell carcinoma.

[0006] 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. Its ligands, PDL1 (programmed cell death 1 ligand 1, also abbreviated as PDL-1) and PDL2 (programmed cell death 1 ligand 2, also abbreviated as PDL-2), both belong to the B7 superfamily, where PDL1 is expressed on many 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.

[0007] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune escape. PD-1 is mainly expressed in immune cells such as T cells, and PDL1, a ligand of PD-1, is mainly highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway activates suppressed T cells and further attacks cancer cells. Blocking the PD-1 / PDL1 signaling promotes the proliferation of tumor antigen-specific T cells, which play a role in killing tumor cells, and further suppresses local tumor growth (Julie R et al., 2012, N Engl J Med .366:2455-2465). Furthermore, tumors with high PDL1 expression 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 the modulation of PD-1 expression by in vivo injection of anti-PD-1 antibodies. Due to the broad antitumor prospects and remarkable efficacy of PD-1 antibodies, antibodies targeting the PD-1 pathway are widely recognized in the industry as a breakthrough in various tumor treatments, such as for the treatment of 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).

[0008] Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is closely related to the CD28 molecule in terms of gene structure, chromosomal location, sequence homology and gene expression. Both are receptors for the costimulatory molecule B7 and are mainly expressed on the surface of activated T cells. Binding of CTLA4 to B7 can inhibit the activation of mouse and human T cells, and it plays a negative regulatory role in T cell activation.

[0009] CTLA4 antibody (or anti-CTLA4 monoclonal antibody) or CTLA4 ligand can inhibit the binding of CTLA4 to its natural ligand, thereby blocking the transmission of negative regulatory signals from CTLA4 to T cells and enhancing the responsiveness of T cells to various antigens. In this respect, the results of in vivo and in vitro studies are basically consistent. CTLA4 monoclonal antibodies are currently in clinical trials or have been approved for use in the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun.13:5.).

[0010] Interleukin 2 (IL-2) is a growth factor produced by T cells that regulates T cell subsets, and is an important factor in regulating immune responses, promoting the proliferation of activated B cells, and participating in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for use in the treatment of malignant tumors (melanoma, renal tumor, etc.), and clinical studies are underway for the treatment of chronic viral infections (Chavez, AR, et al., 2009, Ann NY Acad Sci, 1182: p. 14-27). CTLA4 and CTLA4 antibodies, as important influencing factors of the functional status of T cells, intervene in the body's immune microenvironment. In vitro and in vivo tests have shown that CTLA4 antibodies can specifically release the body's immune suppression caused by CTLA4, activate T cells, and induce the production of IL-2, which has the potential for broad application in anti-tumor and gene therapy of diseases such as parasites.

[0011] In summary, the development of less toxic and more effective treatments and combination treatment strategies would be of great clinical relevance. Summary of the Invention

[0012] As a result of intensive research and creative work, the inventors have expressed recombinant human TIGIT as an antigen using a mammalian cell expression system, immunized mice, and fused mouse spleen cells with myeloma cells to obtain hybridoma cells. The inventors screened a large number of samples to obtain a hybridoma cell line LT019 (deposit number: CTCCC NO: C2020208).

[0013] The present inventors have surprisingly found that the hybridoma cell line LT019 secretes and produces a specific monoclonal antibody (named 26B12) that specifically binds to human TIGIT, and the monoclonal antibody can bind to TIGIT very effectively, reduce the immune cell suppression effect of TIGIT, promote T cell activity, reverse the exhaustion of NK cells, 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, 26B12H4L4).

[0014] The present inventors have also 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.

[0015] This provides the following invention: One aspect of the present invention relates to an anti-TIGIT antibody or 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 antibody comprises HCDR1 to HCDR3 whose amino acid sequences are set forth 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 set forth in SEQ ID NOs: 8 to 10, respectively.

[0016] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof, wherein 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 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.

[0017] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof comprises: 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 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; 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; 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; 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; 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; 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; 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 antibody is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:25.

[0018] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof, wherein the antibody comprises non-CDR regions and the non-CDR regions are derived from a species other than a murine, e.g., a human antibody.

[0019] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof is characterized in that the heavy chain constant region of the antibody is an Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857) and the light chain constant region is an Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0020] 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.

[0021] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof, wherein the antibody has a K of less than 4E-10 or less than 4E-11. D and preferably binds to TIGIT-mFc at the K D is measured with a Fortebio molecular interaction meter.

[0022] 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, 50 is measured by flow cytometry.

[0023] In some embodiments of the invention, the anti-TIGIT antibody is a monoclonal antibody.

[0024] In some embodiments of the invention, the anti-TIGIT antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (eg, a bispecific antibody).

[0025] 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.

[0026] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment is an antibody produced from the hybridoma cell line LT019 deposited at the China Center for Typical Culture Collection (CCTCC) and having the deposit number CCTCC NO: C2020208.

[0027] 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.

[0028] Another aspect of the invention relates to a carrier comprising an isolated nucleic acid molecule of the invention.

[0029] Another aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention or a carrier of the invention.

[0030] Another aspect of the present invention relates to the hybridoma cell line LT019, which was deposited at the China Typical Culture Collection Center (CCTCC) and has the deposit number CTCCC NO: C2020208.

[0031] Another aspect of the present invention relates to a conjugate comprising an antibody and a binding moiety, wherein the antibody is an anti-TIGIT antibody or an antigen-binding fragment thereof described in any one of the present invention, and the binding moiety is a detectable label, preferably, the binding moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0032] Another 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 present invention or comprising a conjugate of the present invention, Preferably, the kit further comprises a second antibody that specifically recognizes 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.

[0033] Another aspect of the present invention relates to the use of an antibody according to any one of the invention or a conjugate of the invention in the manufacture of a kit for detecting the presence or level of TIGIT in a sample.

[0034] Another 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 conjugate of the present invention, optionally further comprising a pharma- ceutically acceptable carrier and / or excipient.

[0035] In one or more embodiments of the invention, the pharmaceutical composition further comprises one or more anti-PD-1 antibodies, or one or more anti-PD-L1 antibodies, such as an anti-CTLA4-anti-PD-1 bispecific antibody.

[0036] In one or more embodiments of the present invention, the pharmaceutical composition comprises the anti-TIGIT antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or anti-PD-L1 antibody, calculated based on the mass of the antibodies, at a mass ratio of (1:5)-(5:1), such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.

[0037] Another aspect of the invention relates to a combination product (e.g., a kit) comprising a first product and a second product, each 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 according to the present invention, or a pharmaceutical composition according to any one of the present invention; the second product comprises at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody, such as an anti-CTLA4-anti-PD-1 bispecific antibody; Preferably, the combination product further comprises a separately packaged third product comprising one or more chemotherapy drugs; Preferably, the first product and the second product further independently comprise one or more pharma- ceutically acceptable auxiliary materials; Preferably, the combination product further comprises a product description.

[0038] In one or more embodiments of the invention, the combination product wherein the mass ratio of the anti-TIGIT antibody or antigen-binding fragment thereof to the anti-PD-1 antibody or anti-PD-L1 antibody, calculated by the mass of the antibodies, is (1:5)-(5:1), for example:1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.

[0039] Another aspect of the present 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 manufacture of a medicament for treating and / or preventing a tumor.

[0040] 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, which is used for treating and / or preventing tumors.

[0041] Another aspect of the present invention relates to a method for treating and / or preventing tumors comprising the step of administering to a subject in need thereof an effective amount of an antibody according to any one of the present invention, a conjugate according to the present invention, a pharmaceutical composition according to any one of the present invention or a combination product according to any one of the present invention.

[0042] The present invention relates to a method for preventing and / or treating a tumor (particularly a malignant tumor) or an infection or infectious disease, the method comprising administering to a subject a therapeutically effective amount of an anti-TIGIT antibody in combination with an anti-CTLA4-anti-PD-1 bispecific antibody, more preferably further in combination with one or more drugs (e.g., chemotherapeutic agents or growth inhibitory agents, targeted therapeutic agents, antibody-drug conjugates, antimetabolite drugs, antibiotics, plant-based anti-cancer drugs and / or hormone-based drugs, adriamycin, tamoxifen, megestrol, and / or asparaginase), preferably, the anti-TIGIT antibody, anti-CTLA4-anti-PD-1 bispecific antibody and tumor chemotherapy drugs are administered simultaneously or sequentially.

[0043] In some embodiments, the chemotherapeutic agent or growth inhibitory agent is selected from platinum-based drugs (e.g., cisplatin, carboplatin, or oxaliplatin), fluorouracil-based antitumor agents, cyclophosphamide, pemetrexed, paclitaxel, vinblastines, adriamycins, goserelin, alkylating agents, anthracene ring systems, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0044] In some embodiments, 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 phosphadiyl inositol 3-kinase inhibitor, an Akt inhibitor, an mTOR inhibitor, a bisphosphadiyl inositol 3-kinase / mTOR inhibitor, and combinations thereof.

[0045] In some embodiments, the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and a radioisotope chelator.

[0046] 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 in combination with an anti-CTLA4-anti-PD-1 bispecific antibody, more preferably further in combination with one or more chemotherapeutic drugs, preferably administering the anti-TIGIT antibody, anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapeutic drugs simultaneously or sequentially.

[0047] In one or more embodiments of the invention, the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, B lymphocytoma, plasma cell carcinoma, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal carcinoma, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, adenocarcinoma (e.g. pancreatic cancer, breast cancer), lung cancer (e.g. small cell lung cancer, small cell lung carcinoma), breast cancer, liver cancer (e.g. hepatocellular carcinoma, hepatobiliary carcinoma), Gastric cancer, gastrointestinal cancer, intestinal cancer (e.g. colon cancer, colorectal cancer), biliary tract cancer (e.g. cholangiocarcinoma), renal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma (e.g. multiple myeloma), non-Hodgkin's lymphoma, B lymphocytoma, plasma cell carcinoma, leukemia, lymphoma, bone cancer, osteosarcoma, chondrosarcoma, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors.

[0048] In one or more embodiments of the invention, the anti-CTLA4-anti-PD-1 bispecific antibody comprises: a first protein functional domain of target PD-1; and comprising a second protein functional domain of target CTLA4, 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; Where: the immunoglobulin comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 27 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 29 to 31, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 28 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 32 to 34, respectively); and the single-chain antibody comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 35 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 37 to 39, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 36 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 40 to 42, respectively); Or, The immunoglobulin comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 35 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 37 to 39, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 36 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 40 to 42, respectively), and the single-chain antibody comprises HCDR1 to HCDR3 in a heavy chain variable region shown in SEQ ID NO: 27 (preferably, HCDR1 to HCDR3 shown in SEQ ID NO: 29 to 31, respectively) and LCDR1 to LCDR3 in a light chain variable region shown in SEQ ID NO: 28 (preferably, LCDR1 to LCDR3 shown in SEQ ID NO: 32 to 34, respectively).

[0049] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, comprising: The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO:27, SEQ ID NO:43, or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 44, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% thereof. or a sequence having at least 99% homology thereto, and the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO:35, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 100%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 149%, at least and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,selected from sequences having at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the Or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO:35, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or a sequence identical to ... and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 36, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 16 and the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from SEQ ID NO: 27, SEQ ID NO: 43, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 100%, at least 150%, at least 105%, at least 150%, at least 15 ... and the amino acid sequence of the light chain variable region of the single chain antibody is selected from SEQ ID NO:28, SEQ ID NO:44, or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,The sequence is selected from sequences having at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology.

[0050] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the bispecific antibody is selected from any one of the following (1) to (20): (1) the amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 28 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 35 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 36 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (2) the amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 28 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 45 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 49 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (3) the amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 28 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 46 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 50 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 44 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 35 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 36 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 44 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 45 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 49 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (6) the amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 43 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 44 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9 ... and the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 46 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147 the amino acid sequence of the light chain variable region of the single chain antibody is represented by SEQ ID NO:50 or a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 35 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 36 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 27 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 28 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 35 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 36 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 43 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 45 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in a sequence having the following structure: SEQ ID NO: 49 or a sequence which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or less identical thereto. The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 27 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 28 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 45 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in a sequence having the following structure: SEQ ID NO: 49 or a sequence which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or less identical thereto. The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 43 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 46 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in a sequence having the following structure: SEQ ID NO: 50 or a sequence which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or less identical thereto. The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 27 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 28 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 46 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in a sequence having the following structure: SEQ ID NO: 50 or a sequence which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or less identical thereto. The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 43 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 28 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 47 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 51 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 28 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 48 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 52 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 44 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 47 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 51 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 43, or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 44 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 48 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 52 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 47 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 51 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 27 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 28 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 48 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO:52 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9 ... and the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 27 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147 the amino acid sequence of the light chain variable region of the single chain antibody is represented by SEQ ID NO:28 or a sequence having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 47 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 51 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 43 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,As shown in a sequence having at least 98% or at least 99% homology, And, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 48 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. and the amino acid sequence of the light chain variable region of said immunoglobulin is set forth in SEQ ID NO: 52 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least The amino acid sequence of the heavy chain variable region of the single chain antibody is represented by SEQ ID NO: 43 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, The amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 44 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%,It is shown in sequences having at least 98% or at least 99% homology.

[0051] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, comprising: The amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO: 53 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. and the amino acid sequence of the light chain is set forth in SEQ ID NO:54 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology thereto.

[0052] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the first protein functional domain and the second protein functional domain are linked directly or via a linking fragment, and / or the heavy chain variable domain of the single chain antibody and the light chain variable domain of the single chain antibody are linked directly or via a linking fragment.

[0053] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the linked fragment is (GGGGS)n, where n is a positive integer, preferably n is 1, 2, 3, 4, 5 or 6.

[0054] In a specific embodiment, the anti-CTLA4-anti-PD-1 bispecific antibody according to the present invention, wherein the first protein functional domain and the second protein functional domain are independently one, two or more than two.

[0055] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the single chain antibody (preferably the heavy chain variable region) is linked to the C-terminus of the immunoglobulin heavy chain.

[0056] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, comprising: the immunoglobulin is a human IgG1 subtype, wherein, according to the EU numbering system, the heavy chain constant region of said immunoglobulin has one of the following mutation combinations: L234A and L235A, or L234A and G237A, or L235A and G237A, or L234A, L235A, G237A.

[0057] In a specific embodiment, the present invention provides an anti-CTLA4-anti-PD-1 bispecific antibody, wherein the bispecific antibody is a first protein functional domain of target PD-1; and comprising a second protein functional domain of target CTLA4, The first protein functional domain is one, and the second protein functional domain is two, wherein the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain antibody; The amino acid sequence of the heavy chain of said immunoglobulin is set forth in SEQ ID NO: 53 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. and the amino acid sequence of the light chain is set forth in SEQ ID NO:54 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; The amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 48 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO:52 or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; The single chain antibody is linked to the C-terminus of an immunoglobulin heavy chain, the first protein functional region and the second protein functional region are linked by a first linking fragment, and the heavy chain variable region of the single chain antibody and the light chain variable region of the single chain antibody are linked by a second linking fragment, the first linking fragment and the second linking fragment being the same or different; Preferably, the amino acid sequences of the first linked fragment and the second linked fragment are independently selected from SEQ ID NO: 55 and SEQ ID NO: 56; Preferably, the amino acid sequences of the first linked fragment and the second linked fragment are both set forth in SEQ ID NO:56.

[0058] In a specific embodiment, the heavy chain amino acid sequence of the anti-CTLA4-anti-PD-1 bispecific antibody according to the present invention is shown in SEQ ID NO:57, the light chain amino acid sequence is shown in SEQ ID NO:59, and the bispecific antibody structure is IgG-scFv, in which the IgG portion is an anti-PD1 antibody and the scFv portion is an anti-CTLA4 antibody; wherein the HCDR1 sequence of the anti-PD1 antibody is set forth in SEQ ID NO: 61, the HCDR2 sequence is set forth in SEQ ID NO: 62, the HCDR3 sequence is set forth in SEQ ID NO: 63, and the VH sequence is set forth in SEQ ID NO: 73; the LCDR1 sequence of the anti-PD1 antibody is set forth in SEQ ID NO: 70, the LCDR2 sequence is set forth in SEQ ID NO: 71, the LCDR3 sequence is set forth in SEQ ID NO: 72, and the VL sequence is set forth in SEQ ID NO: 76; The HCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 64, the HCDR2 sequence is shown in SEQ ID NO: 65, the HCDR3 sequence is shown in SEQ ID NO: 66, the VH sequence is shown in SEQ ID NO: 74, the LCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 67, the LCDR2 sequence is shown in SEQ ID NO: 68, the LCDR3 sequence is shown in SEQ ID NO: 69, and the VL sequence is shown in SEQ ID NO: 75.

[0059] In another aspect of the present invention, the present invention relates to a unit formulation for treating a tumor, preferably comprising 1 to 10,000 mg (preferably, 10 to 1,000 mg, preferably, 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg) of an anti-TIGIT antibody according to any one of the aspects of the present invention, 1 to 10,000 mg (preferably, 1 to 1,000 mg, preferably, 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of an anti-CTLA4-anti-PD-1 bispecific antibody according to any one of the aspects of the present invention, and optionally one or more chemotherapy drugs according to the present invention (e.g., a platinum-based drug and / or a fluorouracil-based antitumor drug), wherein the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and the chemotherapy drug are each packaged separately.

[0060] 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, preferably wherein the anti-CTLA4-anti-PD-1 bispecific antibody, the anti-TIGIT antibody and the chemotherapy drug in said unit formulation are each administered separately.

[0061] In another embodiment of the present invention, preferably, the present invention relates to a single drug dosage unit for treating a tumor, which comprises 0.1 to 10,000 mg (preferably, 1 to 1,000 mg, preferably, 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of an anti-TIGIT antibody according to any one of the present invention and 0.1 to 10,000 mg (preferably, 1 to 1,000 mg, preferably, 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg) of an anti-CTLA4-anti-PD-1 bispecific antibody according to any one of the present invention.

[0062] In one or more embodiments of the present invention, the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and / or the chemotherapy drug are suitable for intravenous injection or infusion, and are preferably in liquid form.

[0063] In one or more embodiments of the present invention, the step of administering to the subject an effective amount of an anti-TIGIT antibody described in any one of the embodiments of the present invention and / or an anti-CTLA4-anti-PD-1 bispecific antibody described in any one of the embodiments of the present invention is before or after surgical treatment and / or before or after radiation treatment.

[0064] In one or more embodiments of the present invention, the single dose of the anti-TIGIT antibody according to any one of the present invention and / or the anti-CTLA4 anti-PD-1 bispecific antibody according to any one of the present invention is 0.1 to 100 mg per kilogram of body weight, preferably 1 to 10 mg, or the single dose of the anti-TIGIT antibody according to any one of the present invention and / or the anti-CTLA4 anti-PD-1 bispecific antibody according to any one of the present invention is 10 to 1000 mg per subject, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg, Preferably, the administration is twice daily to about once every other day, or once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks; Preferably, the mode of administration is intravenous infusion or injection.

[0065] The variable regions of the light and heavy chains determine antigen binding, and each chain variable region contains three highly variable regions called complementarity determining regions (CDRs), the heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and the light chain (L) CDRs include LCDR1, LCDR2, and LCDR3, which are 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 methods for determining antibody CDR regions when the antibody heavy and light chain variable region sequences are known, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, each application of the definition of the CDRs of various antibodies or variants thereof is within the scope of the term as defined and used herein. Given the variable region amino acid sequence of the antibody, one of skill in the art can generally determine particular CDRs without reliance on any experimental data other than the sequence itself.

[0066] 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.

[0067] The amino acid sequence of the CDR region of the monoclonal antibody sequence is analyzed by technical means well known to those skilled in the art, for example, by analysis based on the IMGT definition via the VBASE2 database.

[0068] Antibodies 26B12, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 according to the invention have the same CDRs: 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).

[0069] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all common procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0070] As used herein, when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length of TIGIT protein, or the extracellular immunoglobulin variable region (IgV) structural domain, or a fragment containing the extracellular immunoglobulin variable region (IgV) structural domain. It also includes fusion proteins of TIGIT, such as fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or alterations (including but not limited to substitutions, deletions and / or additions) may be naturally produced or artificially introduced into the amino acid sequence of TIGIT protein without affecting its biological function. Thus, 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. Furthermore, when describing a sequence fragment of TIGIT protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.

[0071] In this specification, the term EC 50 refers to the concentration for 50% of maximal effect, which is the concentration that can produce 50% of the maximum effect.

[0072] As used herein, the term "antibody" refers to an immunoglobulin molecule that typically consists of two pairs of peptides, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, and epsilon, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 3 or more amino acids. Each heavy chain contains a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C H1 , C H2 , C H3Each 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 structural domain, C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. H and V L The regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conservative regions, 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 carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L) each form an antigen-binding site. The assignment of amino acids to each region or structural domain is based on the definitions in 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, as defined by 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 intended to be limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, polyclonal antibodies, among others. The antibody may be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0073] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, which is also referred to as an "antigen-binding portion." See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical destruction 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, diabody antibodies, and such polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0074] 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, i.e., the V domain of a single arm of an antibody. L and V H The term "dAb fragment" refers to an antibody fragment consisting of the V 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 disulfides in the hinge region.

[0075] In some cases, the antigen-binding fragment of an antibody is a single chain antibody (e.g., scFv), wherein V L and V HThe structural domains pair to form monovalent molecules by linkers that can produce them 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 have the general structure NH2-V L -Linker-V H -COOH or NH2-V H -Joint-V L The junction may have a repeating GGGGS amino acid sequence or a variant thereof. For example, a junction having the amino acid sequence (GGGGS)4 may be used, but also variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other junctions that may be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8: 725-731; Choi et al. (2001), Eur. J. Immunol. 31: 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. 94-106.

[0076] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody, in which V H and V L The structural domains are expressed on a single polypeptide chain, but by using short linkers that do not allow pairing between two structural domains on the same chain, the structural domains can pair with complementary structural domains on another chain and create two antigen-binding sites (see, 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)).

[0077] In other cases, the antigen-binding fragment of an antibody is a "bispecific antibody", which refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analogue through a binding arm, and the manner of binding includes, but is not limited to, chemical reaction, gene fusion, and enzymatic reaction. The antigen-binding fragment of an antibody may be a "multispecific antibody", which includes, for example, a trispecific antibody, which is an antibody with three different antigen-binding specificities, and a tetraspecific antibody, which is an antibody with four different antigen-binding specificities. For example, a DARPin is linked to an IgG antibody, scFv-Fc antibody fragment, or a combination thereof, such as CN104341529A. An anti-IL-17a fynomer is linked to an anti-IL-6R antibody, such as WO2015141862A1.

[0078] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 provided herein) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical disruption methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as for intact antibodies.

[0079] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody fragment derived from a population of highly homologous antibody molecules, i.e., completely identical except for possible naturally occurring mutations. Monoclonal antibodies are highly specific to a single epitope on an antigen. In contrast to monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies that recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported 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 can also be obtained by employing recombinant DNA technology (see, for example, US Patent 4, 816, 567).

[0080] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be an antibody of non-human origin (e.g., mouse, rat or rabbit) having the expected specificity, affinity or reactivity. Furthermore, some of the amino acid residues in the framework region (FR) of the acceptor antibody may also be replaced with the corresponding amino acid residues of an antibody of non-human origin or of another antibody to further improve or optimize the properties of the antibody. For details of 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.

[0081] As used herein, the term "isolated" or "isolated" refers to something that has been artificially obtained from a natural state. When a substance or component that is "isolated" in nature appears, the natural environment in which it exists has been altered, or the substance has been isolated from its natural environment, or both have occurred together. For example, a non-isolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide is isolated in high purity from this natural state and is referred to as "isolated". The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0082] As used herein, the term "vector" refers to a nucleic acid carrier into which a polyribonucleotide can be inserted. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs), phages such as lambda and M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). A vector can contain multiple elements that control expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. A vector can also contain a replication origin.

[0083] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK293 cells, or human cells.

[0084] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one that binds to an antigen within about 10 -5Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 Affinity (K D ) means that the antibody binds to the antigen.

[0085] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to express 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. Typically, antibodies have a dissociation equilibrium constant of about 10 -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (K D ) to bind to an antigen (e.g., TIGIT protein). D can be measured using methods known to those of skill in the art, such as using a Fortebio Molecular Interaction Meter.

[0086] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably, and the terms "polyclonal antibody" and "polyclonal antibody" can be used interchangeably. The terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably. The terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Furthermore, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0087] As used herein, the term "pharmaceutically acceptable vector and / or excipient" refers to a vector and / or excipient that is pharma- ceutically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, and the like. For example, pH adjusting agents include, but are not limited to, phosphate buffers, and the like. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancing agents include, but are not limited to, sodium chloride.

[0088] As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a tumor). An effective amount for treating a disease refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.

[0089] As used herein, the terms "hybridoma" and "hybridoma cell line" can be used interchangeably, and when the terms "hybridoma" and "hybridoma cell line" are referred to, subclones and progeny of the hybridoma are also included.

[0090] The term "single drug dosage unit" refers to a single drug dosage form comprising an anti-CTLA4 anti-PD-1 bispecific antibody, anti-TIGIT antibody according to the present invention, administered to a subject at the time of administration (preferably per kg of subject body weight), for example an injection, in an ampoule. In a specific embodiment of the present invention, the administration regimen includes administering the single drug dosage unit according to an administration cycle, for example, twice a day to once about every other day, or once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

[0091] In the present invention, unless otherwise specified, the terms "first" (e.g., first protein functional domain, first linked fragment) and "second" (e.g., second protein functional domain, second linked fragment) are used for the purpose of distinguishing notation or clarifying expression, and do not have a typical sequential meaning.

[0092] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of drug that, alone or in combination with other therapeutic agents, protects a subject from the onset of disease or promotes the elimination of disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free stages of the disease, or the prevention of damage or dysfunction due to the disease affliction. A number of methods known to the skilled practitioner can be used to assess the ability of a therapeutic agent to promote regression of 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.

[0093] A "prophylactically effective amount" of a drug is any amount of drug that inhibits the onset or recurrence of cancer when administered alone or in combination with an anti-tumor agent to a subject at risk of developing cancer (e.g., a subject with pre-disease symptoms) or at risk of cancer recurrence. In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of cancer. "Inhibiting" the onset or recurrence of cancer means reducing the likelihood of the onset or recurrence of cancer, or completely preventing the onset or recurrence of cancer.

[0094] Beneficial Effects of the Invention The monoclonal antibody of the present invention can specifically bind to TIGIT, has extremely strong affinity, and can reduce the inhibitory effect of TIGIT on immune cells, promote the activity of T cells, reverse the depletion of NK cells, and enhance the killing effect of immune cells on tumors. Anti-TIGIT antibody alone or in combination with anti-CTLA4-anti-PD-1 bispecific antibody (and / or chemotherapeutic drug) can effectively treat or prevent tumors, and the combination of anti-TIGIT antibody and anti-CTLA4-anti-PD-1 bispecific antibody has a pharmacological effect of effectively inhibiting tumor growth, which is superior to anti-TIGIT antibody alone or anti-CTLA4-anti-PD-1 bispecific antibody alone, and has good application prospects and market value. [Brief description of the drawings]

[0095] [Figure 1] 1 shows the results of detecting the binding activity of 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, and RG6058 antibodies to TIGIT-mFc. [Diagram 2] 1 shows the results of detecting the binding activity of 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4, and RG6058 antibodies to TIGIT-mFc. [Diagram 3] The results show that 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, and RG6058 antibodies compete with human CD155-hFc-Biotin for binding to TIGIT-mFc. [Figure 4] The results show that 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4, and RG6058 antibodies compete with human CD155-hFc-Biotin for binding to TIGIT-mFc. [Diagram 5] 1 shows the results of affinity constant determination between 26B12H3L3 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 6] 1 shows the results of affinity constant determination between 26B12H1L1 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 7] 1 shows the results of affinity constant determination between 26B12H2L2 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 8] 1 shows the results of affinity constant determination between 26B12H2L3 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 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 constant between 26B12H3L2 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 10] 1 shows the results of affinity constant determination between 26B12H4L4 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 11] 1 shows the results of affinity constant determination between 26B12H1L4 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 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 constant between 26B12H4L1 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 13] 1 shows the results of affinity constant determination between RG6058 and TIGIT-mFc. The antibody concentrations added to the curves from top to bottom in the figure are 5 nM, 1.67 nM, 0.557 nM, 0.185 nM, and 0.06 nM, respectively. [Figure 14] FACS detects the binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT. [Figure 15] FACS detects 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] FACS detects 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] The amount of IL-2 secreted after adding TIGIT antibody to Jurkat-TIGIT and HT1080-aCD3scFv cell lines is detected. [Figure 18] Effect on hTIGIT-BALB / c transgenic mouse CT26 tumor model. [Figure 19] Body weight change in the hTIGIT-BALB / c transgenic mouse CT26 tumor model. [Figure 20] Effect of the combination of 26B12H2L2 and the anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM) on the BALB / c-hPD1 / hTIGIT transgenic mouse CT26 tumor model. [Figure 21] Body weight change in BALB / c-hPD1 / hTIGIT transgenic mouse CT26 tumor model by combination of 26B12H2L2 and anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM). Biological materials related to deposit: The hybridoma cell line LT019 (TIGIT-26B12) was deposited at China Center for Typical Culture Collection (CCTCC) on October 22, 2020, with the deposit number CTCCC NO: C2020208, deposited at Wuhan University, Wuhan, China, zip code 430072. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] The present invention will be described in detail with reference to the following examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting 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 art (for example, see J. Sambrook et al., Molecular Cloning Experimental Guidelines, Third Edition, Science Press, translated by Huang Pedang et al.) or the product instructions. The reagents and instruments used are conventional products available on the market unless the manufacturer is specified. For example, 293T can be purchased from ATCC.

[0097] In the following examples of the present invention, the BALB / c mice used were purchased from Guangdong Medical Experimental Animal Center.

[0098] In the following examples of the present invention, the positive control antibody RG6058 used has a sequence as set forth in SEQ ID NO: 34 and SEQ ID NO: 36 of Chinese Patent Disclosure CN108290946.

[0099] In the following examples of the present invention, the combined anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM) used is derived from an antibody produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., whose sequence is described in the published patent CN112300286A, with reference to the CP004 (hG1TM) heavy chain full length sequence (amino acid sequence shown in SEQ ID NO: 57, nucleotide sequence shown in SEQ ID NO: 58) and light chain full length sequence (amino acid sequence shown in SEQ ID NO: 59, nucleotide sequence shown in SEQ ID NO: 60). The structure of CP004 (hG1TM) is IgG-scFv, of which the IgG part is an anti-PD1 antibody and the scFv part is an anti-CTLA4 antibody;

[0100] wherein the HCDR1 sequence of the anti-PD1 antibody is set forth in SEQ ID NO: 61, the HCDR2 sequence is set forth in SEQ ID NO: 62, the HCDR3 sequence is set forth in SEQ ID NO: 63, the VH sequence is set forth in SEQ ID NO: 73, the LCDR1 sequence of the anti-PD1 antibody is set forth in SEQ ID NO: 70, the LCDR2 sequence is set forth in SEQ ID NO: 71, the LCDR3 sequence is set forth in SEQ ID NO: 72, and the VL sequence is set forth in SEQ ID NO: 76;

[0101] The HCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 64, the HCDR2 sequence is shown in SEQ ID NO: 65, the HCDR3 sequence is shown in SEQ ID NO: 66, the VH sequence is shown in SEQ ID NO: 74, the LCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 67, the LCDR2 sequence is shown in SEQ ID NO: 68, the LCDR3 sequence is shown in SEQ ID NO: 69, and the VL sequence is shown in SEQ ID NO: 75.

[0102] Example 1: Preparation of anti-TIGIT antibody 26B12 1. Preparation of Hybridoma Cell Line LT019 The antigen used in the preparation of anti-TIGIT antibodies was human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2, the sequence of mFc is shown in SEQ ID NO: 77). Spleen cells were collected from immunized mice 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 an antibody that specifically binds to TIGIT. A stable hybridoma cell line was obtained from the hybridoma cells obtained by screening by the finite dilution method. The above hybridoma cell line was named hybridoma cell line LT019, and the monoclonal antibody secreted therefrom was named 26B12.

[0103] The hybridoma cell line LT019 (also called TIGIT-26B12) was deposited at the China Center for Typical Culture Collection (CCTCC) on October 22, 2020, with the deposit number CTCCC NO: C2020208, and deposited at Wuhan University, Wuhan, China, with the postal code 430072.

[0104] 2. Preparation of Anti-TIGIT Antibody 26B12 The LT019 cell line prepared above was cultured in CD medium (Chemical Defined Medium, containing 1% penicillin-streptomycin) under conditions of 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, centrifuged at high speed, vacuum filtered through a micropore filtration membrane, and purified using a HiTrap protein A HP column to produce antibody 26B12.

[0105] Example 2: Sequence analysis of anti-TIGIT antibody 26B12 From the LT019 cell line cultured in Example 1, mRNA was extracted according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, product number DP430).

[0106] cDNA was synthesized and amplified by PCR according to the instructions in the Invitrogen SuperScript(R) III First-Strand Synthesis System for RT-PCR kit.

[0107] The PCR amplification products were directly subjected to TA cloning, and the specific procedures were carried out by referring to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0108] The TA cloning product was directly sequenced, and the sequencing results are as follows: The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:2 and the fragment length is 363 bp.

[0109] The amino acid sequence encoded thereby is shown in SEQ ID NO:1 and is 121 amino acids in length.

[0110] Here, the sequence of heavy chain HCDR1 is shown in SEQ ID NO:3, the sequence of HCDR2 is shown in SEQ ID NO:4 and the sequence of HCDR3 is shown in SEQ ID NO:5.

[0111] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:7 and is 321 bp in length.

[0112] The amino acid sequence encoded thereby is shown in SEQ ID NO:6 and is 107 amino acids in length.

[0113] Here, the sequence of light chain LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9 and the sequence of LCDR3 is shown in SEQ ID NO:10.

[0114] Example 3: Design and preparation of the light and heavy chains of humanized anti-human TIGIT antibodies 1. Design of light and heavy chains of anti-human TIGIT humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained in Example 2, an antibody model was simulated on 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: P01857, and the light chain constant regions were Ig kappa chain C region, ACCESSION: P01834).

[0115] The sequences of the designed variable regions are shown in Table A below. [Table 1]

[0116] Heavy and light chain variable region sequences of humanized monoclonal antibody 26B12H1L4 The nucleic acid sequence of the heavy chain variable region of the above eight antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 is 363 bp in length and the encoded amino acid sequence is 121 aa in length, while the nucleic acid sequence of the light chain variable region is 321 bp in length and the encoded amino acid sequence is 107 aa in length.

[0117] The above eight types of antibodies have the same HCDR1 to HCDR3 and LCDR1 to LCDR3 as follows: The sequence of HCDR1 is shown in SEQ ID NO:3, the sequence of HCDR2 is shown in SEQ ID NO:4 and the sequence of HCDR3 is shown in SEQ ID NO:5.

[0118] The sequence of LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.

[0119] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 The heavy chain constant regions all use the Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant regions all use the Ig kappa chain C region, ACCESSION: P01834.

[0120] 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 cloned into pUC57simple (provided by GenScript) vector, and the resulting vectors were named pUC57simple-26B12H1, pUC57simple-26B12L1; pUC57simple-26B12 pUC57simple-26B12H1, pUC57simple-26B12L4; 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, respectively. Referring to the standard techniques introduced in the "Guidelines for Molecular Cloning Experiments (2nd Edition)", the full-length heavy and light chain genes were digested with EcoRI and HindIII enzymes and subcloned into the expression vector pcDNA3.1 by restriction enzyme (EcoRI and HindIII) digestion 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. Furthermore, the heavy and light chain genes of the recombinant expression plasmids were sequenced and analyzed.Next, the corresponding combinations containing the light chain and heavy chain recombinant plasmid design genes (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, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H4 ...1, NA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4 and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4) were co-transfected into 293F cells, and the culture medium was collected and purified. After the sequencing was verified to be correct, an endotoxin-free expression plasmid was prepared, and the plasmid was instantly transfected into HEK293 cells to express the antibody. After culturing for 7 days, the cell culture medium was collected and affinity purified using a Protein A column to obtain the humanized antibody.

[0121] Example 4: Measuring the binding activity of antibodies to the antigen TIGIT-mFc using ELISA Experimental procedure: 2 μg / mL sheep anti-mouse IgG Fc (purchased from Jackson, lot number: 132560) was coated on a microplate and incubated at 4 ° C for 16 hours. After incubation, the sheep anti-mouse IgG Fc-coated microplate was washed once with PBST, and then blocked for 2 hours using a PBST solution of 1% BSA as a microplate blocking solution. After blocking of the microplate, the plate was washed three times with PBST. Then, 1 μg / mL of the antigen human TIGIT-mFc was added, incubated at 37 ° C for 30 min, and the plate was washed three times with PBST. The antibody gradient-diluted with PBST solution was added to the wells of the microplate, and the antibody dilution gradient is detailed in Tables 1 and 2. The microplate to which the antibody to be measured was added was incubated at 37 ° C for 30 min, and after incubation was completed, the plate was washed three times with PBST. After washing the plate, a working solution of HRP-labeled sheep anti-human IgG Fc (purchased from Jackson, lot number: 128332) secondary antibody diluted at a ratio of 1:5000 was added and incubated at 37°C for 30 min. After incubation was completed, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added to develop the color for 4 min away from light, and the color reaction was stopped by adding a stop solution. The microplate was immediately placed in a microplate reader, a light wavelength of 450 nm was selected, and the OD value of each well of the microplate was read. The data was analyzed and processed with SoftMax Pro 6.2.1 software.

[0122] The results of the 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 abscissa and the absorbance value on the ordinate to determine the EC 50 The results are shown in Tables 1 and 2 and Figures 1 and 2. [Table 2] TIFF2024528682000003.tif56170 [Table 3]

[0123] As a result, it was shown that the antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 could all effectively bind to human TIGIT-mFc, the binding efficiency was dose-dependent, and the binding activity was equivalent to that of RG6058, a positive drug for the same target, and 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 have the function of effectively binding to TIGIT.

[0124] Example 5: Measuring the ability of antibodies to compete with CD155-hFc-Biotin for binding to TIGIT-mFc using a competitive ELISA method Experimental procedure: 2 μg / mL TIGIT-mFc was coated onto a microplate and then incubated overnight at 4°C. After incubation, the antigen-coated microplate was washed once with PBST and then blocked for 2 hours using a 1% BSA in PBST solution as a microplate blocking solution. After blocking of the microplate, the plate was washed three times with PBST. Gradient dilutions of antibodies in PBST solution were added to the microplate, and the antibody concentrations are detailed in Tables 3 and 4. After 10 min incubation at room temperature, an equal volume of 2 μg / mL (final concentration 1 μg / mL) CD155-hFc-Biotin (manufactured by Zhongshan Kangfang Biological Pharmaceutical Co., Ltd., lot number: 20170210, where the GenBank number of CD155 is NP_006496.4, and the sequence of hFc is shown in SEQ ID NO: 78) was added and mixed well with the antibody, and the microplate was incubated at 37 ° C for 30 min. After the 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 incubated at 37 ° C for 30 min. After the incubation was completed, it was washed four times with PBST, and TMB (Neogen, 308177) was added to develop color for 5 min in the dark, and the color development reaction was terminated by adding a stop solution. The microplate was immediately placed in a microplate reader, a light wavelength of 450 nm was selected, and the OD value of each well of the microplate was read. Data were analyzed and processed with SoftMax Pro 6.2.1 software.

[0125] The activity results of the antibodies competing with CD155-hFc-Biotin for binding to TIGIT-mFc are shown in Tables 3 and 4. Curve fitting was performed with the antibody concentration as the abscissa and the absorbance value as the ordinate to determine the EC 50 was calculated, and the results are shown in Tables 3 and 4 and Figures 3 and 4. [Table 4] TIFF2024528682000006.tif64170 [Table 5] TIFF2024528682000008.tif64170

[0126] The results showed that under the same experimental conditions, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 could compete with CD155-hFc-Biotin for binding to the antigen TIGIT-mFc, respectively, and the activity was comparable to that of the same target positive drug RG6058, suggesting that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 had the ability to effectively compete with CD155-hFc-Biotin for binding to TIGIT-mFc.

[0127] Example 6: Measuring the kinetic parameters of binding of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1 and RG6058 to antigen TIGIT-mFc on a Fortebio molecular interaction meter The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. TIGIT-mFc was immobilized on the AMC sensor at a concentration of 3 μg / mL for 50 s, the sensor was equilibrated in the buffer for 60 s, the TIGIT-mFc immobilized on the sensor was bound to the antibody at a concentration of 0.06-5 nM (3-fold dilution) for 120 s, and the protein was dissociated in the buffer for 300 s. The sensor was regenerated using a 10 mM glycine, pH = 1.7 solution. The detection temperature was 37 °C, the detection frequency was 0.3 Hz, and the vibration speed of the sample plate was 1000 rpm. The data was analyzed by fitting with a 1:1 model to obtain the affinity constants.

[0128] The measurement results of the affinity constant between the humanized antibody (as a control antibody) and TIGIT are shown in Table 5, and the detection results are shown in Figures 5 to 13. [Table 6]

[0129] K D is the affinity constant; K D =kdis / kon.

[0130] As a result, the affinity constants of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1, and RG6058 for TIGIT-mFc were 9.64E-11M, 1.64E-11M, 8.40E-12M, 4.85E-11M, 5.40E-11M, 3.69E-11M, 4.63E-11M, 8.57E-12M, and 3.16E-11M, respectively.

[0131] The results showed that the binding affinity of each TIGIT antibody to TIGIT-mFc was, from strongest to weakest, 26B12H2L2, 26B12H4L1, 26B12H1L1, RG6058, 26B12H4L4, 26B12H1L4, 26B12H2L3, 26B12H3L2, and 26B12H3L3. Here, the affinity of the humanized antibodies 26B12H2L2, 26B12H4L1, and 26B12H1L1 was stronger than that of the positive drug RG6058, while the affinity of 26B12H4L4 was equivalent to that of the positive drug RG6058.

[0132] Example 7: FACS detects the binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT Experimental method: TIGIT vector plenti6.3-TIGITFL-BSD (TIGIT is Genbank ID: NP_776160.2, GenScript was commissioned to synthesize the full-length human TIGIT cDNA sequence through gene optimization, named TIGITFL, and cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-TIGITFL plasmid. The TIGITFL target gene fragment was retrieved from the pUC57simple-TIGITFL plasmid synthesized by double enzymes BamHI & XhoI, and subcloned into the plenti6.3 expression vector via the restriction sites BamHI & XhoI. The vector pLenti6.3 was purchased from Invitrogen) was transfected into 293T cells, and the cell line 293T-TIGIT cells, which stably express TIGIT, were obtained by screening.

[0133] 293T-TIGIT cells (DMEM+10%FBS) were collected, centrifuged for 5min to remove the supernatant, resuspended, counted and viability (P7, 95.79%) measured, cells were diluted, 30w cells were added to each well of a clear tip-bottom 96-well plate, 200μL of 1% PBSA was added to each tube, centrifuged for 5min, and the supernatant was removed. 100μL of antibodies (final concentrations 300nM, 100nM, 33.3nM, 11.1nM, 3.7nM, 1.23nM, 0.41nM, 0.041nM, 0.0041nM) were added correspondingly to each well according to the experimental design, blank control and isotype control were designed, and incubated on ice for 60min. 200μL of 1% PBSA was added to each tube, centrifuged for 5min to remove the supernatant, and washed twice. FITC sheep anti-human IgG antibody (purchased from Jackson, lot number: 109-095-098) (diluted 500-fold with PBSA) was added to each sample and incubated on ice for 40 min under light-shielded conditions. 200 μL of PBSA was added to each tube, centrifuged for 5 min, and the supernatant was removed. 200 μL of PBSA was added to resuspend the cells, transferred to a flow tube, and the average fluorescence intensity of the cells at each concentration was detected by flow cytometry. [Table 7]

[0134] The experimental results are shown in Table 6 and FIG. 14. The binding EC of the positive control antibody RG6058 to the cell membrane surface antigen TIGIT 50 The binding EC value of humanized antibody 26B12H2L2 to the cell membrane surface antigen TIGIT was 1.257 nM. 50 was 0.917nM.

[0135] The experimental results show that the binding ability of the humanized antibody 26B12H2L2 to the cell membrane surface antigen TIGIT is stronger than that of the positive control antibody RG6058.

[0136] Example 8: FACS detects the ability of humanized antibodies 26B12H2L2 and RG6058 to compete with CD155 or CD112 for binding to the 293T-TIGIT cell membrane surface antigen TIGIT Experimental method: 293T-TIGIT cells were harvested, centrifuged for 5 min to remove the supernatant, resuspended, counted and viability (94.95%) measured, cells were diluted, 30w cells were added to each well of a clear tip-bottom 96-well plate, 200μL of 1% PBSA was added to each tube, centrifuged for 5 min, and the supernatant was removed. 100μL of antibodies (final concentrations 300nM, 100nM, 33.3nM, 11.1nM, 3.7nM, 1.23nM, 0.123nM, 0.0123nM) were added to each well correspondingly according to the experimental design, blank control and isotype control were designed, and incubated on ice for 30min. CD155 ((final concentration: 10 nM) manufactured by Zhongshan Yasufang Biomedical Co., Ltd., lot number: 20190726, GenBank number of CD155: NP_006496.4) or CD112 ((final concentration: 30 nM) manufactured by Zhongshan Yasufang Biomedical Co., Ltd., lot number: 20190726, GenBank number of CD112: NP_001036189.1) was added to each sample and incubated on ice under light-shielded conditions for 40 min. Furthermore, 200 μL of 1% PBSA was added to each tube, centrifuged for 5 min, the supernatant was removed, and the tube was washed twice. APC sheep anti-mouse IgG (purchased from Biolegend, lot number: 405308) (minimal x-reactivity) antibody (diluted 300 times with PBSA) was added to each sample and incubated on ice under light-shielded conditions 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 in 200 μL of PBSA and transferred to a flow tube, and the mean fluorescence intensity of the cells at each concentration was detected by flow cytometry.

[0137] The experimental results are shown in Table 7 and Figure 15, and Table 8 and Figure 16, respectively. [Table 8] [Table 9]

[0138] The positive control antibody RG6058 competed with CD155 to bind to TIGIT in ECs. 50 The EC binding of humanized antibody 26B12H2L2 to TIGIT by competing with CD155 was 1.212 nM. 50 The positive control antibody RG6058 competed with CD112 to bind to TIGIT. 50 The EC binding of humanized antibody 26B12H2L2 to TIGIT by competing with CD112 was 1.224 nM. 50 was 1.140 nM.

[0139] The results show that the ability of the humanized antibody 26B12H2L2 to bind to the cell membrane surface antigen TIGIT in competition with CD155 or CD112 is stronger than that of the positive control antibody.

[0140] Example 9: Mixed lymphocyte reaction of Jurkat-TIGIT and HT1080-aCD3scFv cell lines with TIGIT antibody Testing Method: The TIGIT vector plenti6.3 / V5-TIGITFL-BSD (vector pLenti6.3 was purchased from Invitrogen) was transfected into Jurkat cells, and the cells were screened to obtain a cell line, Jurkat-TIGIT cells, that stably expresses TIGIT. The anti-CD3 antibody vector pCDH-aCD3scFv-puro was entrusted to GenScript, which optimized the gene and synthesized the cDNA sequence of anti-CD3scFv. The cDNA sequence was then cloned into the pUC57simple (provided by GenScript) vector to obtain the pUC57simple-anti-CD3scFv plasmid. The anti-CD3scFv target gene fragment was recovered from the pUC57simple-anti-CD3scFv plasmid synthesized by XboI&BamHI double enzymes, and subcloned into the pCDH-CMV-MCS-EF1-Puro (purchased from Yubao Biological Co., Ltd.) expression vector via the restriction sites XboI&BamHI (anti-CD3scFv sequence is from reference: Eukaryotic expression of anti-CD3 single chain Fv antibody gene and the characterization of its bioactivities JOURNAL Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 20 (5), 552-555 (2004), PUBMED15367345). It was transfected into HT-1080 cells, and screening was performed to obtain the cell line HT1080-aCD3scFv cells that stably express anti-CD3scFv.

[0141] Jurkat-TIGIT and HT1080-aCD3scFv cells in the logarithmic growth phase were collected in a 96-well plate, and 5W cells were added per well for Jurkat-TIGIT and 1W cells were added per well for HT1080-aCD3scFv. Diluted antibodies (final concentrations 10nM, 50nM, 250nM) were added, followed by anti-human CD28 antibody (purchased from R&D, lot number: MAB342-500) (3μg / mL), and the cells were cultured in an incubator for 48h. The culture supernatant was collected, and the IL-2 content was detected using an IL-2 ELISA detection kit.

[0142] The experimental results are shown in FIG.

[0143] As a result, both humanized antibody 26B12H2L2 and the positive control antibody RG6058 were able to promote IL-2 secretion in the system, and the IL-2 secretion levels of humanized antibody 26B12H2L2 and RG6058 were equivalent at each concentration (10 nM, 50 nM, 250 nM).

[0144] The results show that humanized antibody 26B12H2L2 and the positive control antibody RG6058 have comparable abilities to induce cells to secrete IL-2.

[0145] Example 10: Therapeutic effect of 26B12H2L2 on inoculation of CT26 mouse-implanted tumors in hTigit-BALB / c transgenic mice hTigit-BALB / c transgenic mice (mice purchased from Jiangsu Jisu Yaokang Biotechnology Co., Ltd., the normal TIGIT gene of the purchased transgenic mice was replaced with human TIGIT gene) were inoculated with 500,000 CT26 cells (mouse colon cancer cell line, purchased from ATCC) on the back. The specific procedure of the experiment was to establish a mouse tumor model by inoculating 25 million / ml CT26 cells into mice at 200μl per mouse. There were eight experimental mice in each group, which were divided into an isotype control group (dosage 20mg / kg, administration method intraperitoneal injection (ip), twice a week) and an experimental group (dosage 20mg / kg, administration method intraperitoneal injection (ip), twice a week). The specific scheme is shown in Table 9. [Table 10]

[0146] The experimental results are shown in FIG.

[0147] The results show that 26B12H2L2 significantly reduced tumor volume in the hTIGIT-BALB / c transgenic mouse CT26 tumor model.

[0148] The results demonstrated that 26B12H2L2 had potent therapeutic efficacy in the hTIGIT-BALB / c transgenic mouse CT26 tumor model and may potentially be used for the treatment and / or prevention of tumors, especially colon cancer.

[0149] Furthermore, as shown in FIG. 19, 26B12H2L2 did not affect the body weight of hTIGIT-BALB / c transgenic mice used as a CT26 tumor model, demonstrating that the 26B12H2L2 antibody did not cause toxic side effects in mice.

[0150] Example 11: Effective treatment with anti-TIGIT antibody in combination with anti-CTLA4-anti-PD-1 bispecific antibody To detect the in vivo tumor suppression activity of anti-TIGIT antibody in combination with anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM), CT26 cells (human colon cancer cells, purchased from Jiangsu Jiaxing Pharmaceutical Co., Ltd.) were first inoculated subcutaneously into 5-7 week-old female BALB / c-hPD1 / hTIGIT mice (purchased from Jiangsu Jiaxing Pharmaceutical Co., Ltd.) until the average tumor volume was 80-120 mm. 3 When the tumor volume reached 1000 mg / kg, the mice were randomly divided into 4 groups of 6 mice each according to the tumor volume. The day of grouping was designated as D0, and drug administration began on the day of grouping, D0. For the administration method of the co-administration group, the drugs were prepared individually and administered separately in sequence (there was no particular requirement for the order or interval of administration, and one drug was administered after the other drug). The modeling and specific administration method are shown in Table 10. After administration, the length and width of the tumor in each group were measured, and the tumor volume was calculated. [Table 11]

[0151] The results are shown in Figure 20. As a result, compared with the isotype control antibody hIgG, both CP004(hG1TM) and 26B12H2L2 could effectively suppress the growth of mouse tumors, and the CP004(hG1TM)+26B12H2L2 group showed a combined antitumor effect in this model, and the combined tumor suppression effect was superior to that of the test drug alone group.

[0152] Furthermore, as shown in FIG. 21, the tumor-bearing mice tolerated well the test drugs CP004 (hG1TM) and 26B12H2L2 both administered alone and in combination, and each group had no effect on the body weight of the tumor-bearing mice.

[0153] Although the specific embodiments of the present invention have been described in detail, those skilled in the art can understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all of these modifications are within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and their equivalents.

Chem.

Claims

1. A pharmaceutical composition or kit comprising an anti-TIGIT antibody or its antigen-binding fragment and an anti-CTLA4-anti-PD-1 bispecific antibody or its antigen-binding fragment, The pharmaceutical composition may further, if necessary, include a pharmaceutically acceptable carrier and / or excipient. The anti-TIGIT antibody comprises HCDR1 to HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 1 and LCDR1 to LCDR3 contained in the light chain variable region shown in SEQ ID NO: 6 (preferably, according to the IMGT numbering system, the antibody comprises a heavy chain variable region containing HCDR1 to HCDR3 having the amino acid sequences shown in SEQ ID NOs: 3 to 5 and a light chain variable region containing LCDR1 to LCDR3 having the amino acid sequences shown in SEQ ID NOs: 8 to 10). The aforementioned anti-CTLA4-anti-PD-1 bispecific antibody is The first protein functional domain that targets PD-1 and The second protein functional domain that targets CTLA4 and Includes, Here, 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. Here, The immunoglobulin comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 27 (preferably HCDR1 to HCDR3 shown in SEQ ID NOs: 29 to 31, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 28 (preferably LCDR1 to LCDR3 shown in SEQ ID NOs: 32 to 34, respectively), and the single-chain antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 35 (preferably HCDR1 to HCDR3 shown in SEQ ID NOs: 37 to 39, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 36 (preferably LCDR1 to LCDR3 shown in SEQ ID NOs: 40 to 42, respectively), Or, The immunoglobulin comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 35 (preferably HCDR1 to HCDR3 shown in SEQ ID NOs: 37 to 39, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 36 (preferably LCDR1 to LCDR3 shown in SEQ ID NOs: 40 to 42, respectively), and the single-chain antibody comprises HCDR1 to HCDR3 in the heavy chain variable region shown in SEQ ID NO: 27 (preferably HCDR1 to HCDR3 shown in SEQ ID NOs: 29 to 31, respectively) and LCDR1 to LCDR3 in the light chain variable region shown in SEQ ID NO: 28 (preferably LCDR1 to LCDR3 shown in SEQ ID NOs: 32 to 34, respectively). Pharmaceutical composition or kit.

2. 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. Preferably, with respect to the anti-TIGIT antibody or its antigen-binding fragment, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 6, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 19, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 19, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 21, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 23, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 21, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 23, The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 25, or The heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:

25. Preferably, the anti-TIGIT antibody or its antigen-binding fragment includes a non-CDR region derived from a species other than a murine, for example, a human antibody. Preferably, the anti-TIGIT antibody or its antigen-binding fragment comprises a heavy chain constant region which is the Ig gamma-1 chain C region (e.g., NCBI deposit: P01857) and a light chain constant region which is the Ig kappa chain C region (e.g., NCBI deposit: P01834), Preferably, the anti-TIGIT antibody or its antigen-binding fragment is Fab, Fab', F(ab') ２ Selected from Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, humanized antibodies, chimeric antibodies, or bispecific antibodies, Preferably, the anti-TIGIT antibody or its antigen-binding fragment has a K content of less than 4E-10 or less than 4E-11. Ｄ It is bonded to TIGIT-mFc, preferably the K Ｄ This was measured using the Fortebio molecular interaction instrument. Preferably, the anti-TIGIT antibody or its antigen-binding fragment has an EC of less than 1.5 nM, less than 1.2 nM, or less than 1 nM. ５０ It binds to TIGIT-mFc, preferably the EC ５０ This is measured by a flow cytometer. Preferably, the anti-TIGIT antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., a bispecific antibody). Preferably, the antigen-binding fragment is Fab, Fab', F(ab') ２ Selected from Fd, Fv, dAb, Fab / c, complementarity-determining region fragments, single-chain antibodies (e.g., scFv), humanized antibodies, chimeric antibodies, or bispecific antibodies, Preferably, the anti-TIGIT antibody or its antigen-binding fragment is an antibody produced by the hybridoma cell line LT019 deposited with the China Typical Culture Depository Center (CCTCC) under depositary number C2020208. The pharmaceutical composition or kit according to claim 1.

3. Regarding the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NOs. 27 and 43, or sequences having at least 80% homology thereto; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NOs. 28 and 44, or sequences having at least 80% homology thereto; the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NOs. 35, 45, 46, 47 and 48, or sequences having at least 80% homology thereto; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NOs. 36, 49, 50, 51 and 52, or sequences having at least 80% homology thereto; Or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NOs. 35, 45, 46, 47, and 48, or sequences having at least 80% homology thereto; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NOs. 36, 49, 50, 51, and 52, or sequences having at least 80% homology thereto; the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NOs. 27 and 43, or sequences having at least 80% homology thereto; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NOs. 28 and 44, or sequences having at least 80% homology thereto; Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the bispecific antibody is one of the following (1) to (20): (1) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto; (2) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 45 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 49 or a sequence having at least 80% homology thereto; (3) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto; (4) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto; (5) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 45 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 49 or a sequence having at least 80% homology thereto; (6) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto; (7) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto; (8) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto; (9) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 45 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 49 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto; (10) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 45 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 49 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto; (11) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto; (12) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto; (13) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 47 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 51 or a sequence having at least 80% homology thereto; (14) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto; (15) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 47 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 51 or a sequence having at least 80% homology thereto; (16) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto; (17) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 47 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 51 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto; (18) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 27 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 28 or a sequence having at least 80% homology thereto; (19) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 47 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 51 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto; And, (20) The heavy chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, the light chain variable region of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto, the heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 43 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody is shown in the amino acid sequence shown in SEQ ID NO: 44 or a sequence having at least 80% homology thereto; One of the following will be selected: Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 53 or a sequence having at least 80% homology thereto, and the light chain has the amino acid sequence shown in SEQ ID NO: 54 or a sequence having at least 80% homology thereto. Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the first protein functional region is linked to the second protein functional region either directly or via a linking fragment, and / or the heavy chain variable region of the single-chain antibody is linked to the light chain variable region of the single-chain antibody either directly or via a linking fragment. Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the linking fragment is (GGGGS)n, where n is a positive integer, preferably n is 1, 2, 3, 4, 5, or 6. Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the number of the first protein functional region and the second protein functional region is one, two, or more, independently of each other. Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the single-chain antibody (preferably the heavy chain variable region) is ligated to the C-terminus of the heavy chain of the immunoglobulin. Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the immunoglobulin is of the human IgG1 subtype. Here, according to the EU numbering system, the heavy chain constant region of the immunoglobulin is determined by the following combination of mutations: L234A and L235A, or L234A and G237A, or L235A and G237A, or L234A, L235A, and G237A Having one of the following, Preferably, with respect to the anti-CTLA4-anti-PD-1 bispecific antibody, the bispecific antibody is The first protein functional domain targeting PD-1, The second protein functional domain that targets CTLA4 and Includes, Here, the number of the first protein functional regions is one, and the number of the second protein functional regions is two. The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody. The heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 53 or a sequence having at least 80% homology thereto, and the light chain has the amino acid sequence shown in SEQ ID NO: 54 or a sequence having at least 80% homology thereto. The heavy chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the light chain variable region of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto. The single-chain antibody is ligated to the C-terminus of the heavy chain of the immunoglobulin, The first protein functional region is linked to the second protein functional region via a first linking fragment, 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 either identical or different. Preferably, the first linkage fragment and the second linkage fragment each have an amino acid sequence independently selected from SEQ ID NO: 55 and SEQ ID NO: 56, Preferably, the amino acid sequences of the first linkage fragment and the second linkage fragment are shown in SEQ ID NO:

56. Preferably, the heavy chain of the anti-CTLA4-anti-PD-1 bispecific antibody has the amino acid sequence shown in SEQ ID NO: 57, and its light chain has the amino acid sequence shown in SEQ ID NO: 59, and the bispecific antibody has an IgG-scFv structure, where the IgG portion is an anti-PD1 antibody and the scFv portion is an anti-CTLA4 antibody. Here, the HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO: 61, the HCDR2 sequence is shown in SEQ ID NO: 62, the HCDR3 sequence is shown in SEQ ID NO: 63, the VH sequence is shown in SEQ ID NO: 73, the LCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO: 70, the LCDR2 sequence is shown in SEQ ID NO: 71, the LCDR3 sequence is shown in SEQ ID NO: 72, and the VL sequence is shown in SEQ ID NO:

76. The HCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 64, the HCDR2 sequence is shown in SEQ ID NO: 65, the HCDR3 sequence is shown in SEQ ID NO: 66, the VH sequence is shown in SEQ ID NO: 74, the LCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO: 67, the LCDR2 sequence is shown in SEQ ID NO: 68, the LCDR3 sequence is shown in SEQ ID NO: 69, and the VL sequence is shown in SEQ ID NO:

75. The pharmaceutical composition or kit according to claim 1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the anti-TIGIT antibody or its antigen-binding fragment and the anti-CTLA4-anti-PD-1 antibody or its antigen-binding fragment are present in an antibody-based mass ratio 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.

5. A kit comprising individually packaged first and second products, The first product comprises an anti-TIGIT antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3. The second product comprises an anti-CTLA4-anti-PD-1 bispecific antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 3. Preferably, the kit further comprises individually packaged third-party products containing one or more chemotherapy drugs. Preferably, the first product and the second product further independently comprise one or more pharmaceutically acceptable adjuvants. Preferably, the product of the combination further includes an accompanying document. Preferably, in the kit, the anti-TIGIT antibody or its antigen-binding fragment and the anti-CTLA4-anti-PD-1 bispecific antibody or its antigen-binding fragment are present in an antibody-based mass ratio 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. kit.

6. Use of an anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 1 to 3 and an anti-CTLA4-anti-PD-1 bispecific antibody or its antigen-binding fragment as defined in any one of claims 1 to 3 for the manufacture of a pharmaceutical, kit, composition, unit formulation or single-dose unit for the treatment and / or prevention of tumors, Preferably, the pharmaceutical, kit, composition, unit formulation or single-dose unit comprises one or more drugs (e.g., chemotherapeutic agents or growth inhibitors, targeted therapeutic agents, antibody-drug conjugates, antimetabolites, antibiotics, antihormone agents, plant-derived anticancer agents, and / or hormonal drugs), where preferably, the drugs include the following: adriamycin, tamoxifen, megestrol, asparaginase, platinum-based drugs (e.g., cisplatin, carboplatin, or oxaliplatin), fluorouracil anticancer agents, cyclophosphamide, pemetrexed, paclitaxel, vinca alkaloids, adriamycin, goserelin, alkylating agents, anthracite One or more of the following are selected: Clin, antiandrogens, aromatase inhibitors, protein kinase inhibitors (e.g., tyrosine kinase inhibitors), lipid kinase inhibitors, antisense oligonucleotides, ribozymes, topoisomerase inhibitors, cytotoxic agents, antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, retinoids, histone deacetylase inhibitors, B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors, mytansine, monomethyl auristatin E, calicheamicin, esperamycin, and radioisotope chelating agents. Preferably, the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and the antitumor chemotherapy drug are administered simultaneously or sequentially, and more preferably, the anti-TIGIT antibody and the anti-CTLA4-anti-PD-1 bispecific antibody are administered before or after surgical procedures and / or before or after radiotherapy. Preferably, the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and / or the chemotherapy drug are in a form suitable for intravenous injection or intravenous infusion, preferably in liquid form. Preferably, the tumor is as follows: Breast cancer, ovarian cancer, colorectal cancer, cervical tumors, multiple myeloma, non-Hodgkin lymphoma, B lymphoma, plasma cell carcinoma, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, adenocarcinoma (e.g., pancreatic cancer), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), breast cancer, liver cancer (e.g., hepatocellular carcinoma and hepatobiliary tract cancer), stomach cancer, gastrointestinal cancer, intestinal cancer (e.g., colon cancer and colorectal cancer), Biliary tract cancer (e.g., cholangiocarcinoma), kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma (e.g., multiple myeloma), non-Hodgkin lymphoma, B lymphoma, plasma cell carcinoma, leukemia, lymphoma, bone cancer, osteosarcoma, chondrosarcoma, and solid tumors with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), Selected from one or more of the following: Preferably, the unit dose of the anti-TIGIT antibody and / or anti-CTLA4 anti-PD-1 bispecific antibody as defined in any one of claims 1 to 3 is 0.1 to 100 mg, preferably 1 to 10 mg, per kilogram of body weight, or the unit dose of the anti-TIGIT antibody and / or anti-CTLA4 anti-PD-1 bispecific antibody as described in any one of claims 1 to 3 is 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg for each subject. Preferably, the above dose is administered twice a day to approximately once every two days, or once every three days, four days, five days, six days, ten days, one week, two weeks, three weeks, four weeks, five weeks, or six weeks. Preferably, the route of administration is intravenous infusion or intravenous injection. use.

7. An anti-TIGIT antibody as defined in any one of claims 1 to 3, in an amount of 1 to 10,000 mg (preferably 10 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg), An anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 3, in an amount of 1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg), and If necessary, one or more drugs (e.g., chemotherapeutic agents or growth inhibitors, targeted therapies, antibody-drug conjugates, antimetabolites, antibiotics, antihormone agents, plant-derived anticancer agents, and / or hormonal drugs) A unit formulation comprising, preferably a unit formulation used to treat a tumor, Preferably, the drug is one of the following: adriamycin, tamoxifen, megestrol, asparaginase, platinum-based drugs (e.g., cisplatin, carboplatin or oxaliplatin), fluorouracil anticancer drugs, cyclophosphamide, pemetrexed, paclitaxel, vinca alkaloids, adriamycin, goserelin, alkylating agents, anthracyclines, antiandrogens, aromatase inhibitors, protein kinase inhibitors (e.g., tyrosine kinase inhibitors), lipid kinase inhibitors, One or more of the following are selected: antisense oligonucleotides, ribozymes, topoisomerase inhibitors, cytotoxic agents, antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, retinoids, histone deacetylase inhibitors, B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, dual phosphatidylinositol 3-kinase / mTOR inhibitors, mytansine, monomethyl auristatin E, calicheamicin, esperamycin, and radioisotope chelating agents. The anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and the chemotherapy drug are packaged separately. Unit formulation.

8. An anti-TIGIT antibody as defined in any one of claims 1 to 3, in an amount of 0.1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg), and An anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 to 3, in an amount of 0.1 to 10,000 mg (preferably 1 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, 200 mg, or 100 mg). including, A single dose unit, preferably a single dose unit used to treat a tumor.

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