Pharmaceutical compositions and uses

Abstract

The present application belongs to the field of medicine, 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. Specifically, the present application relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 3-5 respectively; and the light chain variable region of the antibody comprises LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 8-10 respectively. The antibody of the present application has the potential of being applied to tumor prevention and treatment, alone or in combination with the bispecific antibody.

Description

Technical Field

[0001] This 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 (T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9) is a member of the poliovirus receptor (PVR) / Nectin family. TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type I transmembrane domain, and an intracellular domain with classical immunoreceptor tyrosine inhibitory motif (ITIM) and immunoglobulin tyrosine tail (ITT) motifs. TIGIT is highly expressed in lymphocytes, especially 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 / nectin 2), and CD113 (also known as PVRL3) are ligands that bind to TIGIT (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors[J]. Nature Reviews Immunology, 2015, 15(4):243-254), among which 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 on TIGIT-high expression 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). Reports have shown that simultaneous blocking of PD-1 and TIGIT can 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]. Cancercell, 2014, 26(6):923-937). Recent studies have found that TIGIT, as an immune checkpoint for NK cells, can lead to NK cell exhaustion during tumor development, and have demonstrated that anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and be used in the immunotherapy of various tumors (Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J]. Nature immunology, 2018, 19(7):723-732).

[0004] Furthermore, reports indicate that TIGIT inhibitors, alone or in combination with PD-1 inhibitors and CD96 inhibitors, significantly reduce 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 inhibitors, alone or in combination with TIGIT inhibitors and / or PD-1 inhibitors, increase the ability of TILs to produce cytokines in ovarian tumors, endometrial tumors, 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, as novel immune checkpoint antibody drugs, have broad application prospects and can be used for tumor immunotherapy. Roche's Tiragolumab is in phase 3 clinical trials, and it has been reported that the combination of Tiragolumab monoclonal antibody and PD-L1 drug Tecentriq (atezolizumab) as first-line therapy in a phase 2 clinical study of patients with PD-L1 positive metastatic non-small cell lung cancer (NSCLC) showed that the combination of Tiragolumab and Tecentriq was well tolerated, with a 43% reduction in the risk of disease progression, and the combination therapy was effective (Exit C. Roche to present first clinical data on novel anti-TIGIT cancer immunotherapy tiragolumab at ASCO[J]). Existing clinical data indicate 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-cell 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 in activated T cells, B cells, and myeloid cells. PD-1 ligands PDL1 (Programmed cell death 1 ligand 1, also known as PDL-1) and PDL2 (Programmed cell death 1 ligand 2, also known as PDL-2) both belong to the B7 superfamily. PDL1 is expressed in various cell types, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only in antigen-presenting cells such as dendritic cells and macrophages.

[0007] The PD-1 / PDL1 signaling pathway plays a crucial role in regulating immune tolerance, microbial infection, and tumor immune escape. PD-1 is primarily expressed on immune cells such as T cells, while its ligand, PDL1, is highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate suppressed T cells, which then attack cancer cells. Blocking PD-1 / PDL1 signaling can also promote the proliferation of tumor antigen-specific T cells, which kill tumor cells and thus inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366:2455–2465). Furthermore, tumors with high PDL1 expression are often associated with difficult-to-detect cancers (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104:3360-5). One effective approach is to regulate PD-1 expression through in vivo injection of anti-PD-1 antibodies. Due to the broad-spectrum anti-tumor prospects and remarkable efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring about breakthroughs in the treatment of various tumors: for example, 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) and CD28 molecules share very similarities in gene structure, chromosomal location, sequence homology, and gene expression. Both are receptors for the co-stimulatory molecule B7 and are primarily expressed on the surface of activated T cells. CTLA4 binding to B7 inhibits the activation of mouse and human T cells, playing a negative regulatory role in T cell activation.

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

[0010] Interleukin-2 (IL-2), produced by T cells, is a growth factor regulating T cell subsets and an important factor in regulating immune responses. It also promotes the proliferation of activated B cells and participates in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors (including melanoma and renal tumors) and is currently undergoing clinical trials 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 T cell function, intervene in the body's immune microenvironment. In in vitro and in vivo experiments, CTLA4 antibodies can specifically relieve the immunosuppression caused by CTLA4, activate T cells, and induce IL-2 production, showing broad application prospects in gene therapy for diseases such as tumors and parasites.

[0011] In conclusion, developing less toxic and more effective treatments, as well as combination therapy regimens, has significant clinical implications. Summary of the Invention

[0012] Through in-depth research and creative work, the inventors used a mammalian cell expression system to express recombinant human TIGIT as an antigen to immunize mice. Hybridoma cells were then obtained by fusing mouse spleen cells with myeloma cells. Through screening a large number of samples, the inventors obtained the hybridoma cell line LT019 (accession number CCTCC NO: C2020208).

[0013] The inventors have surprisingly discovered that the hybridoma cell line LT019 can secrete specific monoclonal antibodies (named 26B12) that specifically bind to human TIGIT. These monoclonal antibodies effectively bind to TIGIT, reducing its inhibitory effect on immune cells, promoting T cell activity, reversing NK cell depletion, and enhancing the tumor-killing effect of immune cells. Furthermore, the inventors have creatively synthesized humanized antibodies against human TIGIT (named 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4).

[0014] The inventors also surprisingly discovered that the antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 of the present invention have TIGIT binding activity and extremely strong affinity; 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 can effectively reduce TIGIT activity.

[0015] This leads to the following invention:

[0016] One aspect of the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, wherein,

[0017] The anti-TIGIT antibody comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:1 and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:6.

[0018] Preferably, according to the IMGT numbering system, the heavy chain variable region of the antibody contains amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs:3-5; and the light chain variable region of the antibody contains amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NOs:8-10.

[0019] In one or more embodiments of the present invention, the anti-TIGIT antibody or its 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

[0020] The amino acid sequence of the variable region of the light chain 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.

[0021] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment, wherein...

[0022] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.

[0023] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:19.

[0024] 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:19.

[0025] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:21.

[0026] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:23.

[0027] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:21.

[0028] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:23.

[0029] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:25; or

[0030] 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.

[0031] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment thereof, wherein the antibody includes a non-CDR region and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0032] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment thereof, wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857); and the light chain constant region is Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0033] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or biantibody.

[0034] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment, wherein the antibody has a K value of less than 4E-10 or less than 4E-11. D Combined with TIGIT-mFc; preferably, the K D Measured using a Fortebio molecular interaction analyzer.

[0035] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment, wherein the antibody is in EC50 at a concentration of less than 1.5 nM, less than 1.2 nM, or less than 1 nM. 50 Combined with TIGIT-mFc; preferably, the EC 50 Measured by flow cytometry.

[0036] In some embodiments of the present invention, the anti-TIGIT antibody is a monoclonal antibody.

[0037] In some embodiments of the present invention, the anti-TIGIT antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0038] In some embodiments of the present invention, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, complementarity-determining region fragment, single-chain antibody (e.g., scFv), humanized antibody, chimeric antibody, or bispecific antibody.

[0039] In one or more embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment thereof, wherein the antibody is an antibody produced by the hybridoma cell line LT019, the hybridoma cell line LT019 being deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:C2020208.

[0040] Another aspect of the invention relates to isolated nucleic acid molecules that encode the anti-TIGIT antibody or its antigen-binding fragment as described in any one of the invention.

[0041] Another aspect of the invention relates to a carrier comprising the isolated nucleic acid molecules of the invention.

[0042] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecules of the invention, or the vector of the invention.

[0043] Another aspect of the present invention relates to the hybridoma cell line LT019, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:C2020208.

[0044] Another aspect of the present invention relates to a conjugate comprising an antibody and a conjugation portion, wherein the antibody is any one of the anti-TIGIT antibodies or its antigen-binding fragments as described in the present invention, and the conjugation portion is a detectable label; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0045] Another aspect of the present invention relates to a kit comprising any anti-TIGIT antibody or antigen-binding fragment thereof as described in any one of the present invention, or comprising a conjugate of the present invention;

[0046] Preferably, the kit further includes a second antibody that specifically recognizes the antibody; optionally, the second antibody further includes a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0047] Another aspect of the invention relates to the use of any antibody or conjugate of the invention as described in any one of the invention in the preparation of a kit for detecting the presence or level of TIGIT in a sample.

[0048] Another aspect of the invention relates to a pharmaceutical composition comprising any one of the anti-TIGIT antibodies or antigen-binding fragments thereof, or conjugates thereof, as described in any one of the invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0049] In one or more embodiments of the present invention, the pharmaceutical composition further comprises one or more anti-PD-1 antibodies, or one or more anti-PD-L1 antibodies, such as anti-CTLA4-anti-PD-1 bispecific antibodies.

[0050] In one or more embodiments of the present invention, the pharmaceutical composition wherein, based on the mass of the antibody, the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody 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.

[0051] Another aspect of the invention relates to a combination product (e.g., a reagent kit) comprising individually packaged first and second products, wherein,

[0052] The first product comprises any one of the anti-TIGIT antibodies or antigen-binding fragments thereof, conjugates of the present invention, or pharmaceutical compositions of the present invention as described in any one of the present invention;

[0053] The second product contains 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;

[0054] Preferably, the combined product further comprises an individually packaged third product, the third product containing one or more chemotherapy drugs.

[0055] Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients.

[0056] Preferably, the combined product further includes a product instruction manual.

[0057] In one or more embodiments of the present invention, the combined product wherein, based on the mass of the antibody, the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody 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.

[0058] Another aspect of the present invention relates to the use of any antibody, conjugate, pharmaceutical composition, or combination product described in any one of the present invention in the preparation of a medicament for treating and / or preventing tumors.

[0059] The antibody, conjugate, pharmaceutical composition, or combination product according to any one of the present invention is used for the treatment and / or prevention of tumors.

[0060] Another aspect of the invention relates to a method of treating and / or preventing tumors, comprising the step of administering to a subject in need an effective amount of any of the antibodies, conjugates, pharmaceutical compositions, or combination products of the invention as described in any of the invention.

[0061] This invention relates to a method for preventing and / or treating tumors (particularly malignant tumors) or infections or infectious diseases, comprising administering a therapeutically effective amount of an anti-TIGIT antibody to a subject, in combination with an anti-CTLA4-anti-PD-1 bispecific antibody, more preferably, further in combination with one or more drugs (such as chemotherapeutic agents or growth inhibitors, targeted therapies, antibody-drug conjugates, antimetabolites, antibiotics, herbal anticancer drugs and / or hormonal drugs, doxorubicins, tamoxifen, megestrol acetate, and / or asparaginases), preferably, the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the tumor chemotherapeutic drug are administered simultaneously or sequentially.

[0062] In some embodiments, the chemotherapeutic agent or growth inhibitor is selected from platinum-based drugs (such as cisplatin, carboplatin, or oxaliplatin), fluorouracil antitumor drugs, cyclophosphamide, pemetrexed, paclitaxel, vinca alkaloids, doxorubicins, goserelin, alkylating agents, anthracyclines, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0063] In some embodiments, the targeted therapeutic agent is selected from B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, bisphosphatidylinositol 3-kinase / mTOR inhibitors, and combinations thereof.

[0064] In some embodiments, the antibody-drug conjugate comprises a drug selected from the group consisting of: maydencin, monomethyl auristatin E, galicariin, esperamicin, and a radioisotope chelating agent.

[0065] This invention relates to a method for preventing and / or treating tumors (particularly malignant tumors), comprising administering a therapeutically effective amount of an anti-TIGIT antibody to a subject, in combination with an anti-CTLA4-anti-PD-1 bispecific antibody, more preferably, further in combination with one or more chemotherapeutic agents. Preferably, the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and the chemotherapeutic agents are administered simultaneously or sequentially.

[0066] In one or more embodiments of the present 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-cell lymphoma, plasma cell carcinoma, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, adenocarcinoma (such as pancreatic cancer, breast cancer), lung cancer (such as small cell lung cancer), breast cancer, liver cancer (such as hepatocellular carcinoma, hepatobiliary cancer), and gastric cancer. Gastrointestinal cancer, colorectal cancer (such as colon cancer, colorectal cancer), biliary tract cancer (such as bile duct cancer), kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma (such as multiple myeloma), non-Hodgkin lymphoma, B lymphoma, plasma cell carcinoma, leukemia, lymphoma, bone cancer, osteosarcoma, chondrosarcoma, high microsatellite instability (MSI-H) or mismatch repair deficient (dMMR) solid tumors.

[0067] In one or more embodiments of the present invention, the anti-CTLA4-anti-PD-1 bispecific antibody comprises:

[0068] Targeting the first protein functional region of PD-1, and

[0069] Targeting the second protein functional region of CTLA4;

[0070] 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.

[0071] in,

[0072] The immunoglobulin comprises HCDR1-HCDR3 (preferably HCDR1-HCDR3 as shown in SEQ ID NO:27) in the heavy chain variable region, and LCDR1-LCDR3 (preferably LCDR1-LCDR3 as shown in SEQ ID NO:32-34) in the light chain variable region, as shown in SEQ ID NO:28; and the single-chain antibody comprises HCDR1-HCDR3 (preferably HCDR1-HCDR3 as shown in SEQ ID NO:35) in the heavy chain variable region, and LCDR1-LCDR3 (preferably LCDR1-LCDR3 as shown in SEQ ID NO:36) in the light chain variable region, as shown in SEQ ID NO:40-42.

[0073] or,

[0074] The immunoglobulin comprises HCDR1-HCDR3 (preferably as shown in SEQ ID NOs:37-39) in the heavy chain variable region of SEQ ID NO:36 and LCDR1-LCDR3 (preferably as shown in SEQ ID NOs:40-42) in the light chain variable region of SEQ ID NO:36; and the single-chain antibody comprises HCDR1-HCDR3 (preferably as shown in SEQ ID NOs:29-31) in the heavy chain variable region of SEQ ID NO:27 and LCDR1-LCDR3 (preferably as shown in SEQ ID NOs:32-34) in the light chain variable region of SEQ ID NO:28.

[0075] In a specific implementation scheme, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention, wherein,

[0076] The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:27, SEQ ID NO:43 or sequences 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 with it; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:28, SEQ ID NO:43 or sequences 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 97%, at least 98%, or at least 99% homology with it; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:28, SEQ ID NO:43 or sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 80%, at least 81%, at least 82%, at least 83%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with it; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO:28, SEQ ID NO:43 or sequences having at least 60%, at least 65%, at least 70%, at least 70%, at least 80%, 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%, or at least 99% homology therewith; 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, SEQ ID NO:49 ... 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% homology therewith; 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%, at least 90%, at least 91%, 92%, or 93% homology therewith. Sequences with at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology;

[0077] or,

[0078] The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:35, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or sequences 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 with the sequences thereon; 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:48 or sequences thereon. 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% homology thereto; 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:52 ... 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%, or at least 99% homology therewith; 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%, 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 therewith.

[0079] In a specific implementation, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention is selected from any one of (1)-(20) below:

[0080] (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:35 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 therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:35. 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%, at least 98%, or at least 99% homology as indicated by other sequences.

[0081] (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:45 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 therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:45. 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%, at least 98%, or at least 99% homology with it;

[0082] (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:46. 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%, at least 98%, or at least 99% homology as indicated by other sequences.

[0083] (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:43. 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%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:35 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 therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:35. 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%, at least 98%, or at least 99% homology as indicated by other sequences.

[0084] (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:43. 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%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:45 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 therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:45. 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%, at least 98%, or at least 99% homology with it;

[0085] (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:43. 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%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:46. 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 with it;

[0086] (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:35 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 therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:35. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology with the sequence shown.

[0087] (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:35 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 therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:35. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:43. 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%, or at least 99% homology as indicated by other sequences.

[0088] (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:45 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 therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:45. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology with the sequence shown.

[0089] (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:45 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 therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:45. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:43. 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%, or at least 99% homology as indicated by other sequences.

[0090] (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:46. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology with the sequence shown.

[0091] (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:46. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:43. 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%, or at least 99% homology as indicated by other sequences.

[0092] (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:47 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 therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:47. 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%, at least 98%, or at least 99% homology as indicated by other sequences.

[0093] (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:48. 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% homology with it;

[0094] (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:43. 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%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:47 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 therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:47. 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%, at least 98%, or at least 99% homology as indicated by other sequences.

[0095] (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:43. 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%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:48. 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% homology with it;

[0096] (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:47 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 therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:47. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:27. 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%, at least 98%, or at least 99% homology with the sequence shown.

[0097] (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:48. 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% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:27. 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 with it;

[0098] (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:47 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 therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:47. 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%, at least 98%, or at least 99% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:43. 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%, or at least 99% homology as indicated by other sequences.

[0099] as well as,

[0100] (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown 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% homology therewith, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:48. 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% homology therewith; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in 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%, or at least 99% homology therewith, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:43. 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%, or at least 99% homology.

[0101] In a specific implementation scheme, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention, wherein,

[0102] The heavy chain amino acid sequence of the immunoglobulin is as shown 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% homology therewith, and the light chain amino acid sequence is as shown 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 therewith.

[0103] In a specific implementation, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention, wherein the first protein functional region is directly linked to the second protein functional region or linked through a linker fragment; and / or the heavy chain variable region of the single-chain antibody is directly linked to the light chain variable region of the single-chain antibody or linked through a linker fragment.

[0104] In a specific implementation, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention, wherein the linker fragment is (GGGGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

[0105] In a specific implementation, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention comprises, independently, one, two, or more of the first and second protein functional regions.

[0106] In a specific implementation, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention is wherein the single-chain antibody (preferably the heavy chain variable region) is linked to the C-terminus of the heavy chain of the immunoglobulin.

[0107] In a specific implementation scheme, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention, wherein,

[0108] The immunoglobulin is human IgG1 subtype;

[0109] According to the EU numbering system, the heavy chain constant region of the immunoglobulin has one of the following combinations of mutations:

[0110] L234A and L235A; or

[0111] L234A and G237A; or

[0112] L235A and G237A; or

[0113] L234A, L235A, G237A.

[0114] In a specific implementation, the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention comprises:

[0115] Targeting the first protein functional region of PD-1, and

[0116] Targeting the second protein functional region of CTLA4;

[0117] The first protein has one functional region, and the second protein has two functional regions;

[0118] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0119] The heavy chain amino acid sequence of the immunoglobulin is as shown 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% homology therewith, and the light chain amino acid sequence is as shown 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 therewith;

[0120] The amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown 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% homology therewith; and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown 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% homology therewith.

[0121] The single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin;

[0122] The first protein functional region and the second protein functional region are connected by a first linker fragment; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker fragment; the first linker fragment and the second linker fragment may be the same or different;

[0123] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO:55 and SEQ ID NO:56;

[0124] Preferably, the amino acid sequences of both the first linker fragment and the second linker fragment are as shown in SEQ ID NO:56.

[0125] In a specific embodiment, the heavy chain amino acid sequence of the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention is shown in SEQ ID NOs:57, and the light chain amino acid sequence is shown in SEQ ID NOs:59. The bispecific antibody structure is IgG-scFv, wherein the IgG portion is an anti-PD1 antibody, and the scFv portion is an anti-CTLA4 antibody.

[0126] The HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:61, the HCDR2 sequence in SEQ ID NO:62, the HCDR3 sequence in SEQ ID NO:63, and the VH sequence in SEQ ID NO:73. The LCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:70, the LCDR2 sequence in SEQ ID NO:71, the LCDR3 sequence in SEQ ID NO:72, and the VL sequence in SEQ ID NO:76.

[0127] 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, and 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.

[0128] In another aspect of the invention, a unit formulation is provided, preferably for the treatment of tumors, wherein the unit formulation comprises: 1 to 10,000 mg (preferably 10-1,000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg) of an anti-TIGIT antibody according to any aspect of the invention and 1 to 10,000 mg (preferably 1-1,000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of an anti-CTLA4-anti-PD-1 bispecific antibody according to any aspect of the invention, and optionally one or more chemotherapeutic agents according to the invention (such as platinum-based drugs and / or fluorouracil-based antitumor drugs); wherein the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapeutic agent are each packaged separately.

[0129] This invention relates to a method for the prevention or treatment of cancer or tumors, wherein one or more doses of the unit formulation described herein are administered to a subject in need, preferably wherein the anti-CTLA4-anti-PD-1 bispecific antibody, anti-TIGIT antibody and chemotherapy drug in the unit formulation are administered separately.

[0130] In another aspect of the invention, a single-dose drug unit, preferably for treating tumors, comprises 0.1-10000 mg (preferably 1-1000 mg, more preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of the anti-TIGIT antibody as described in any one of the invention and 0.1-10000 mg (preferably 1-1000 mg, more preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of the anti-CTLA4-anti-PD-1 bispecific antibody as described in any one of the invention.

[0131] In one or more embodiments of the present invention, the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and / or the chemotherapeutic agent are in a form suitable for intravenous injection or intravenous infusion, preferably in liquid form.

[0132] In one or more embodiments of the present invention, the step of administering an effective amount of the anti-TIGIT antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of any one of the present invention to a subject is performed before or after surgical treatment, and / or before or after radiotherapy.

[0133] In one or more embodiments of the present invention, the single dose of the anti-TIGIT antibody and / or the anti-CTLA4 anti-PD-1 bispecific antibody of any one of the present invention is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single dose of the anti-TIGIT antibody and / or the anti-CTLA4 anti-PD-1 bispecific antibody of any one of the present invention is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg.

[0134] Preferably, the medication is administered twice daily to approximately every other day, or once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

[0135] Preferably, the administration method is intravenous infusion or intravenous injection.

[0136] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; named by Kabat et al., see Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242). Given the known sequences of the antibody's heavy and light chain variable regions, several methods exist for determining antibody CDR regions, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of each definition of a CDR for an antibody or its variants will be within the scope of the terminology defined and used herein. Given the amino acid sequence of the antibody's variable region, those skilled in the art can typically determine a particular CDR without relying on any experimental data outside of that sequence itself.

[0137] Preferably, the CDR can also 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.

[0138] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of a monoclonal antibody sequence according to the IMGT definition using the VBASE2 database.

[0139] The antibodies 26B12, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 involved in this invention have the same CDR:

[0140] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0141] HCDR1:GHSFTSDYA (SEQ ID NO:3)

[0142] HCDR2:ISYSDST(SEQ ID NO:4)

[0143] HCDR3:ARLDYGNYGGAMDY(SEQ ID NO:5);

[0144] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0145] LCDR1: QHVSTA (SEQ ID NO:8)

[0146] LCDR2: SAS (SEQ ID NO:9)

[0147] LCDR3: QQHYITPWT (SEQ ID NO: 10).

[0148] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0149] As used herein, when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full-length TIGIT protein, or the extracellular immunoglobulin variable region (IgV) domain, or a fragment containing the extracellular immunoglobulin variable region (IgV) domain; it also includes fusion proteins of TIGIT, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the TIGIT protein without affecting its biological function. Therefore, in this invention, the term "TIGIT protein" or "TIGIT" should include all such sequences, including the sequence shown and its natural or artificial variants. Furthermore, when describing a sequence fragment of the TIGIT protein, it includes not only the sequence fragment but also the corresponding sequence fragment in its natural or artificial variants.

[0150] As used in this article, the term EC 50 The half-maximal concentration (WMC) is the concentration that produces a 50% maximum effect.

[0151] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by 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 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V LThese amino acids form antigen-binding sites. The allocation of amino acids to each region or domain follows the 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 definition of 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. The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0152] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the 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 the antigen; it is also referred to as the “antigen-binding moiety.” See also 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 generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. 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 polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0153] As used in this article, the term "Fd fragment" refers to a segment composed of V H and C H 1. Antibody fragments composed of structural domains; the term "Fv fragment" refers to a fragment consisting of the V domain of a single arm of the antibody. L and V H Antibody fragments composed of structural domains; the term "dAb fragment" refers to an antibody fragment composed of V... H Antibody fragments composed of structural domains (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to a fragment composed of V... L V H C L and C H An antibody fragment consisting of a 1-domain structure; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.

[0154] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where V L and V H The domain enables the formation of monovalent molecules by linker pairing for single polypeptide chains (see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules can have a general structure: NH2-V L -Connector-V H -COOH or NH2-V H -Connector-V L -COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0155] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, where V...H and V L The domain is expressed on a single polypeptide chain, but the linker is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0156] In other cases, the antigen-binding fragment of an antibody is a "bispecific antibody," referring to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analogue through a conjugation arm. The conjugation methods include, but are not limited to, chemical reactions, gene fusion, and enzymatic reactions. The antigen-binding fragment of an antibody can also be a "multispecific antibody," including, for example, trispecific antibodies and tetraspecific antibodies. The former is an antibody with three different antigen-binding specificities, while the latter is an antibody with four different antigen-binding specificities. For example, a designed ankylosing spiculation protein (DARPin) linked to an IgG antibody, an scFv-Fc antibody fragment, or a combination thereof, such as CN104341529A. Anti-IL-17a fynomer binds to an anti-IL-6R antibody, such as WO2015141862A1.

[0157] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0158] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see US Patent 4,816,567).

[0159] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, 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.

[0160] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0161] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0162] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells, or human cells.

[0163] 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 its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0164] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to an antigen (e.g., TIGIT protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0165] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0166] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0167] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease.

[0168] As used herein, the terms “hybridoma” and “hybridoma cell line” are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, they also include subclones and progeny cells of the hybridoma.

[0169] The term "single drug dose unit" refers to a single-dose formulation, such as an injection, containing the anti-CTLA4 anti-PD-1 bispecific antibody and anti-TIGIT antibody described in this invention, to be administered to the subject at the time of the dosing regimen (preferably per kg of the subject's body weight). In specific embodiments of this invention, the dosing regimen includes, for example, administering the single drug dose unit according to a dosing cycle of approximately twice daily to once every other day, or once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

[0170] In this invention, unless otherwise specified, the terms "first" (e.g., first protein functional region, first linker fragment) and "second" (e.g., second protein functional region, second linker fragment) are used for distinguishing reference or for clarity of expression, and do not have a typical meaning of order.

[0171] A “therapeuticly effective amount” or “therapeutic dose” of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of disease or promotes disease remission, demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases, or prevention of damage or disability caused by disease suffering. The ability of a therapeutic agent to promote disease remission can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predicting efficacy for humans, or by determining the activity of the drug in in vitro assays.

[0172] The "preventive effective amount" of a drug refers to any amount of drug, when administered alone or in combination with an antitumor agent to a subject at risk of developing cancer (e.g., a subject with a pre-existing condition) or at risk of cancer recurrence, that inhibits the occurrence or recurrence of cancer. In some embodiments, the preventive effective amount completely prevents the occurrence or recurrence of cancer. "Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of cancer occurring or recurring, or completely preventing the occurrence or recurrence of cancer.

[0173] Beneficial effects of the invention

[0174] The monoclonal antibody of this invention can bind specifically to TIGIT with strong affinity, reducing the inhibitory effect of TIGIT on immune cells, promoting T cell activity, reversing NK cell depletion, and enhancing the tumor-killing effect of immune cells. Anti-TIGIT antibodies, alone or in combination with anti-CTLA4-anti-PD-1 bispecific antibodies (and / or chemotherapy drugs), can effectively treat or prevent tumors. The combination of anti-TIGIT antibodies and anti-CTLA4-anti-PD-1 bispecific antibodies has a pharmacological effect of effectively inhibiting tumor growth, and is superior to either anti-TIGIT antibody monotherapy or anti-CTLA4-anti-PD-1 bispecific antibody monotherapy, showing good application prospects and market value. Attached Figure Description

[0175] Figure 1 Results of the binding activity assay of antibodies 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2 and RG6058 with TIGIT-mFc.

[0176] Figure 2 Results of the binding activity assay of antibodies 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 with TIGIT-mFc.

[0177] Figure 3 The results of the activity assay of antibodies 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2 and RG6058 competing with human CD155-hFc-Biotin for binding to TIGIT-mFc.

[0178] Figure 4 The results of the activity assay of antibodies 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 competing with human CD155-hFc-Biotin for binding to TIGIT-mFc.

[0179] Figure 5 Figure 26B12 H3L3 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0180] Figure 6 Figure 26B12 H1L1 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0181] Figure 7 Figure 26B12 H2L2 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0182] Figure 8 Figure 26B12 H2L3 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0183] Figure 9 Figure 26B12 H3L2 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0184] Figure 10 Figure 26B12 H4L4 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0185] Figure 11 Figure 26B12 H1L4 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0186] Figure 12 Figure 26B12 H4L1 and TIGIT-mFc affinity constant detection results. The antibody concentrations added to each pair of 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.

[0187] Figure 13 Figure 1 shows the results of the affinity constant detection of RG6058 and TIGIT-mFc. The antibody concentrations added to each pair of 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.

[0188] Figure 14 FACS was used to detect the binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT.

[0189] Figure 15 FACS was used to detect the activity of humanized antibodies 26B12H2L2 and RG6058 in competing with CD155 for binding to 293T-TIGIT on the cell membrane surface TIGIT.

[0190] Figure 16 FACS was used to detect the activity of humanized antibodies 26B12H2L2 and RG6058 in competing with CD112 for binding to 293T-TIGIT on the cell membrane surface TIGIT.

[0191] Figure 17 The study aimed to detect the amount of IL-2 secreted after the addition of TIGIT antibody to Jurkat-TIGIT and HT1080-aCD3scFv cell systems.

[0192] Figure 18 Effects of hTIGIT-BALB / c transgenic mouse CT26 tumor model.

[0193] Figure 19 Changes in body weight in hTIGIT-BALB / c transgenic mouse CT26 tumor model.

[0194] Figure 20 The effect of combining 26B12H2L2 with the anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM) on the CT26 tumor model in BALB / c-hPD1 / hTIGIT transgenic mice.

[0195] Figure 21 The combination of 26B12H2L2 and the anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM) was used to investigate the changes in body weight in the CT26 tumor model of BALB / c-hPD1 / hTIGIT transgenic mice.

[0196] Biological materials involved in preservation:

[0197] The hybridoma cell line LT019 (TIGIT-26B12) was deposited at the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with accession number CCTCC NO: C2020208, at Wuhan University, Wuhan, China, 430072, China. Detailed Implementation

[0198] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased on the market. For example, 293T can be purchased from ATCC.

[0199] In the following embodiments of the present invention, the BALB / c mice used were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0200] In the following embodiments of the present invention, the positive control antibody RG6058 used has sequences 34 and 36 as disclosed in Chinese Patent Publication CN108290946.

[0201] In the following embodiments of the present invention, the combined anti-CTLA4-anti-PD-1 bispecific antibody CP004(hG1TM) used was manufactured by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence is derived from the antibody described in the published patent CN112300286A. Referring to the full-length heavy chain sequence of CP004(hG1TM) (amino acid sequence as shown in SEQ ID NOs:57, nucleotide sequence as shown in SEQ ID NO:58) and the full-length light chain sequence (amino acid sequence as shown in SEQ ID NOs:59, nucleotide sequence as shown in SEQ ID NO:60), the structure of CP004(hG1TM) is IgG-scFv, wherein the IgG portion is an anti-PD1 antibody and the scFv portion is an anti-CTLA4 antibody.

[0202] The HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:61, the HCDR2 sequence in SEQ ID NO:62, the HCDR3 sequence in SEQ ID NO:63, and the VH sequence in SEQ ID NO:73. The LCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:70, the LCDR2 sequence in SEQ ID NO:71, the LCDR3 sequence in SEQ ID NO:72, and the VL sequence in SEQ ID NO:76.

[0203] The HCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO:64, the HCDR2 sequence in SEQ ID NO:65, the HCDR3 sequence in SEQ ID NO:66, and the VH sequence in SEQ ID NO:74. The LCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO:67, the LCDR2 sequence in SEQ ID NO:68, the LCDR3 sequence in SEQ ID NO:69, and the VL sequence in SEQ ID NO:75.

[0204] Example 1: Preparation of anti-TIGIT antibody 26B12

[0205] 1. Preparation of hybridoma cell line LT019

[0206] The antigen used to prepare the anti-TIGIT antibody was human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2, and the mFc sequence is shown in SEQ ID NO: 77). Spleen cells from immunized mice were fused with mouse myeloma cells to create hybridoma cells. Using human TIGIT-mFc as the antigen, the hybridoma cells were screened using an indirect ELISA method to obtain hybridoma cells capable of secreting antibodies that specifically bind to TIGIT. The selected hybridoma cells were then subjected to limiting dilution to obtain stable hybridoma cell lines. These hybridoma cell lines were named hybridoma cell line LT019, and the monoclonal antibody secreted by them was named 26B12.

[0207] The hybridoma cell line LT019 (also known as TIGIT-26B12) was deposited at the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with accession number CCTCC NO: C2020208. The deposit address is Wuhan University, Wuhan, China, 430072, China.

[0208] 2. Preparation of anti-TIGIT antibody 26B12

[0209] The LT019 cell line prepared above was cultured in CD medium (Chemical Defined Medium, containing 1% penicillin and streptomycin) at 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, centrifuged at high speed, filtered through a microporous membrane under vacuum, and purified using a HiTrap protein A HP column to obtain antibody 26B12.

[0210] Example 2: Sequence analysis of anti-TIGIT antibody 26B12

[0211] mRNA was extracted from the LT019 cell line cultured in Example 1 according to the method of the Total RNA Extraction Kit for Cultured Bacteria (Tiangen, catalog number DP430).

[0212] According to Invitrogen III. The First-Strand Synthesis System for RT-PCR kit instructions describe the synthesis of cDNA and its PCR amplification.

[0213] The PCR amplification products were directly cloned using TA. For specific procedures, please refer to the instructions for the pEASY-T1 Cloning Kit (TransgenCT101).

[0214] The TA clone product was directly sequenced, and the sequencing results are as follows:

[0215] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, with a fragment length of 363 bp.

[0216] Its encoded amino acid sequence is shown in SEQ ID NO:1, and has a length of 121 amino acids.

[0217] The sequence of the 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.

[0218] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:7, and its length is 321bp.

[0219] Its encoded amino acid sequence is shown in SEQ ID NO:6, and has a length of 107 amino acids.

[0220] The sequence of the 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.

[0221] Example 3: Design and preparation of light and heavy chains of humanized antibodies against human TIGIT

[0222] 1. Light and heavy chain design of humanized anti-human TIGIT antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4.

[0223] 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 by computer, and then mutations were designed according to the model to obtain the variable region sequences (antibody constant region sequences, from the NCBI database; the heavy chain constant region was Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region was Ig kappa chain C region, ACCESSION: P01834).

[0224] The sequence of variable regions in the design is shown in Table A below.

[0225]

[0226] Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 26B12H1L4

[0227] The above eight antibodies, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4, all have a heavy chain variable region nucleic acid sequence length of 363 bp, encoding an amino acid sequence length of 121 aa; and a light chain variable region nucleic acid sequence length of 321 bp, encoding an amino acid sequence length of 107 aa.

[0228] Furthermore, the above 8 antibodies share the same HCDR1-HCDR3 and LCDR1-LCDR3, as follows:

[0229] 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;

[0230] 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.

[0231] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4

[0232] The constant region of the heavy chain was Ig gamma-1 chain C region, ACCESSION: P01857; the constant region of the light chain was Ig kappa chain C region, ACCESSION: P01834.

[0233] The heavy chain cDNA and light chain cDNA of 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, 26B12H2L4, and 26B12H4L4 were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-26B12H1, 26B12H1L4, and 26B12H2L4 vectors, respectively. pUC57simple-26B12L1; pUC57simple-26B12H4, pUC57simple-26B12L1; pUC57simple-26B12H2, pUC57simple-26B12L2; pUC57simple-26B12H3, pUC57simple-26B12L2; pUC57simple-26B12H2, pUC57simple-26B12L3; pUC57simple-26B12H3, pUC57simple-26B12L3; pUC57simple-26B12H1, pUC57simple-26B12L4; pUC57simple-26B12H2, pUC57simple-26B12L4; and pUC57simple-26B12H4 pUC57simple-26B12L4. Following the standard techniques described in *Molecular Cloning: A Laboratory Manual (Second Edition)*, the full-length heavy and light chain genes synthesized by EcoRI and HindIII restriction enzyme digestion were subcloned into the expression vector pcDNA3.1 to obtain expression plasmids pcDNA3.1-26B12H1, pcDNA3.1-26B12L1, pcDNA3.1-26B12H4, pcDNA3.1-26B12H2, pcDNA3.1-26B12L2, pcDNA3.1-26B12H3, pcDNA3.1-26B12L3, and pcDNA3.1-26B12L4. Further sequencing analysis of the heavy / light chain genes in the recombinant expression plasmids was then performed.Subsequently, gene combinations containing the corresponding light and heavy chain recombinant plasmids were designed (pcDNA3.1-26B12H1 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, ...). After co-transfecting 293F cells with pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4 and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4 respectively, the culture medium was collected and purified. After sequencing verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column to obtain humanized antibodies.

[0234] Example 4: ELISA method for determining the binding activity of antibody to antigen TIGIT-mFc

[0235] Experimental Procedure: 2 μg / mL of goat anti-mouse IgG Fc (purchased from Jackson, batch number: 132560) was coated onto the ELISA plate and incubated at 4°C for 16 hours. After incubation, the plate coated with goat anti-mouse IgG Fc was washed once with PBST, followed by blocking with 1% BSA in PBST solution for 2 hours. After blocking, the plate was washed three times with PBST. Then, 1 μg / mL of human TIGIT-mFc was added, and the plate was incubated at 37°C for 30 minutes, followed by washing three times with PBST. Serially diluted antibodies in PBST solution were added to the wells of the ELISA plate (antibody dilution gradients are detailed in Tables 1 and 2). The ELISA plate containing the antibody was incubated at 37°C for 30 minutes, followed by washing three times with PBST. After washing the plate, add HRP-labeled goat anti-human IgG Fc (purchased from Jackson, batch number: 128332) secondary antibody working solution diluted 1:5000 and incubate at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) for color development in the dark for 4 minutes, and add stop solution to terminate the color development reaction. Immediately place the plate in a microplate reader and read the OD values ​​of each well at a wavelength of 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0236] The result of antibody binding to antigen TIGIT-mFc is as follows Figure 1 , Figure 2 As shown in Tables 1 and 2, the OD values ​​for each dose are presented. A curve was fitted with antibody concentration on the x-axis and absorbance value on the y-axis to calculate the EC50 of the antibody-antigen binding. 50 The results are shown in Tables 1 and 2. Figure 1 , Figure 2 As shown.

[0237] Table 1: Binding activity assay results of 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2 and RG6058 with TIGIT-mFc

[0238] Table 2: Binding activity assay results of 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 with TIGIT-mFc

[0239]

[0240]

[0241] The results showed that antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 could effectively bind to human TIGIT-mFc, with binding efficiency showing a dose-dependent relationship. The binding activity was comparable to that of the positive drug RG6058 targeting the same site, indicating that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 have the function of effectively binding TIGIT.

[0242] Example 5: Competitive ELISA method to measure the competitive binding of antibody and CD155-hFc-Biotin to TIGIT-mFc. activity

[0243] Experimental Procedure: TIGIT-mFc was coated onto the ELISA plate at 2 μg / mL and incubated overnight at 4°C. After incubation, the plate coated with antigen was washed once with PBST, followed by blocking with 1% BSA in PBST solution for 2 hours. After blocking, the plate was washed three times with PBST. Serially diluted antibody in PBST solution was added to the ELISA plate (antibody concentrations are detailed in Tables 3 and 4). After incubation at room temperature for 10 minutes, an equal volume of 2 μg / mL (final concentration 1 μg / mL) of CD155-hFc-Biotin (manufactured by Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number: 20170210, where CD155 GenBank number is NP_006496.4, and the hFc sequence is shown in SEQ ID NO:78) was added. The mixture was thoroughly combined with the antibody, and the ELISA plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. After washing the plate, add SA-HRP working solution diluted 1:4000 and incubate at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) for color development in the dark for 5 minutes. Stop the color development reaction by adding stop solution. Immediately place the plate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0244] The results of the antibody's competitive activity against CD155-hFc-Biotin in binding to TIGIT-mFc are shown in Tables 3 and 4. Curve fitting was performed with antibody concentration on the x-axis and absorbance value on the y-axis to calculate the EC50 of the antibody against CD155-hFc-Biotin in competitive binding to TIGIT-mFc. 50 The results are shown in Tables 3 and 4 below. Figure 3 and Figure 4 As shown.

[0245] Table 3: Activity assay results of 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2 and RG6058 competing with CD155-hFc-Biotin for binding to TIGIT-mFc.

[0246] Table 4: Activity assay results of 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 competing with CD155-hFc-Biotin for binding to TIGIT-mFc.

[0247]

[0248] 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, and their activity was comparable to that of the positive drug RG6058 targeting the same site. This suggests that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 have the function of effectively competing with CD155-hFc-Biotin for binding to TIGIT-mFc.

[0249] Example 6: Determination of humanized antibodies 26B12H3L3, 26B12H1L1, and... using a Fortebio molecular interaction analyzer 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1, and RG6058 are related to the antigen. kinetic parameters of TIGIT-mFc combination

[0250] 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 equilibrated in the buffer for 60 s. The immobilized TIGIT-mFc bound to the antibody at a concentration of 0.06–5 nM (three-fold dilution) for 120 s. The protein dissociated in the buffer for 300 s. The sensor was regenerated using 10 mM glycine solution at pH 1.7. The detection temperature was 37°C, the detection frequency was 0.3 Hz, and the sample plate vibration rate was 1000 rpm. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0251] The affinity constants of the humanized antibody (as a control antibody) and TIGIT are shown in Table 5. The detection results are as follows: Figures 5 to 13 As shown.

[0252] Table 5: Detection results of affinity constants between humanized antibodies and TIGIT-mFc antigen

[0253]

[0254] K D K is the affinity constant. D =kdis / kon.

[0255] The results showed that the affinity constants of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1 and RG6058 with 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.

[0256] The results showed that the affinity of each TIGIT antibody for TIGIT-mFc, from strongest to weakest, was as follows: 26B12H2L2, 26B12H4L1, 26B12H1L1, RG6058, 26B12H4L4, 26B12H1L4, 26B12H2L3, 26B12H3L2, and 26B12H3L3. Among these, the humanized antibodies 26B12H2L2, 26B12H4L1, and 26B12H1L1 showed stronger affinity than the positive control RG6058, while the affinity of 26B12H4L4 was comparable to that of the positive control RG6058.

[0257] Example 7: FACS detection of the binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT.

[0258] Experimental methods:

[0259] The TIGIT vector plenti6.3-TIGITFL-BSD (TIGIT is Genbank ID: NP_776160.2; the full-length human TIGIT cDNA sequence was optimized and synthesized by GenScript and named TIGITFL, then cloned into the pUC57simple (provided by GenScript) vector to obtain the pUC57simple-TIGITFL plasmid. The pUC57simple-TIGITFL plasmid synthesized by double digestion with BamHI & XhoI was used to recover the TIGITFL target gene fragment and subcloned it into the plenti6.3 expression vector (pLenti6.3 purchased from Invitrogen) via BamHI & XhoI restriction sites. 293T cells were transfected with this vector, and the stably TIGIT-expressing cell line 293T-TIGIT cells were obtained after selection.

[0260] Collect 293T-TIGIT cells (DMEM + 10% FBS), centrifuge for 5 min, discard the supernatant, resuspend, count and measure viability (P7, 95.79%), dilute the cells, add 30w of cells to each well of a clear conical-bottom 96-well plate, add 200 μL of 1% PBSA to each tube, centrifuge for 5 min, discard the supernatant. Add 100 μL of antibody to each well according to the experimental design (final concentrations: 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM), and design blank controls and isotype controls. Incubate on ice for 60 min. Add 200 μL of 1% PBSA to each tube, centrifuge for 5 min, discard the supernatant, and wash twice. Add FITC goat anti-human IgG antibody (purchased from Jackson, catalog number: 109-095-098) (diluted 500 times with PBSA) to each sample and incubate on ice in the dark for 40 min. Add 200 μL of PBSA to each tube, centrifuge for 5 min, and discard the supernatant. Resuspend the cells in 200 μL of PBSA, transfer to flow cytometry tubes, and detect the average fluorescence intensity of cells at each concentration using flow cytometry.

[0261] Table 6: FACS detection of the binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT.

[0262]

[0263]

[0264] The experimental results are shown in Table 6 and Figure 14 As shown, the positive control antibody RG6058 binds to the cell membrane surface antigen TIGIT in EC. 50 The concentration was 1.257 nM, while the humanized antibody 26B12H2L2 bound to the cell membrane surface antigen TIGIT in EC... 50 It is 0.917 nM.

[0265] Experimental results show that the humanized antibody 26B12H2L2 has a stronger ability to bind to the cell membrane surface antigen TIGIT than the positive control antibody RG6058.

[0266] Example 8: FACS detection of competitive binding of humanized antibodies 26B12H2L2 and RG6058 to CD155 or CD112 Activity of 293T-TIGIT cell membrane surface antigen TIGIT

[0267] Experimental Methods: 293T-TIGIT cells were collected, centrifuged for 5 min, the supernatant was discarded, and the cells were resuspended, counted, and their viability was measured (94.95%). Cells were diluted, and 300 μL of cells were added to each well of a clear conical-bottom 96-well plate. 200 μL of 1% PBSA was added to each well, and the plates were centrifuged for 5 min, then the supernatant was discarded. According to the experimental design, 100 μL of antibody was added to each well (final concentrations: 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM). Blank and isotype controls were also included. Cells were incubated on ice for 30 min. Add either CD155 (final concentration 10 nM) produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number: 20190726, where the GenBank accession number for CD155 is NP_006496.4) or CD112 (final concentration 30 nM) produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., batch number: 20190726, where the GenBank accession number for CD112 is NP_001036189.1) to each sample, incubate on ice in the dark for 60 min, then add 200 μL of 1% PBSA to each tube, centrifuge for 5 min, discard the supernatant, and wash twice. Add APC goat anti-mouse IgG (purchased from Biolegend, batch number 405308) (minimum x-reactivity) antibody (300-fold dilution with PBSA) to each sample, incubate on ice in the dark for 40 min, then add 200 μL of PBSA to each tube, centrifuge for 5 min, and discard the supernatant. Resuspend the cells in 200 μL of PBSA, transfer them to flow cytometry tubes, and detect the average fluorescence intensity of the cells at each concentration using flow cytometry.

[0268] The experimental results are shown in Table 7 and 8 respectively. Figure 15 and Table 8 and Figure 16 As shown.

[0269] Table 7: FACS detection of the activity of humanized antibodies 26B12H2L2 and RG6058 in competitive binding to 293T-TIGIT cell membrane surface antigen TIGIT with CD155.

[0270]

[0271]

[0272] Table 8: FACS detection of the activity of humanized antibodies 26B12H2L2 and RG6058 in competitive binding to 293T-TIGIT cell membrane surface antigen TIGIT with CD112.

[0273]

[0274] The results showed that the positive control antibody RG6058 competitively inhibited CD155 binding to TIGIT on ECG. 50 The concentration was 1.212 nM, while the humanized antibody 26B12H2L2 competed with CD155 for TIGIT binding to ECMO. 50 The concentration was 1.049 nM; the positive control antibody RG6058 competed with CD112 binding to TIGIT in EC. 50 The concentration was 1.224 nM, while the humanized antibody 26B12H2L2 competed with CD112 for TIGIT binding on ECGs. 50 It is 1.140 nM.

[0275] The results showed that the humanized antibody 26B12H2L2 was more competitive than the positive control antibody RG6058 in binding to the cell membrane surface antigen TIGIT via CD155 or CD112.

[0276] Example 9: A mixed lymphocyte culture with TIGIT antibody added to the Jurkat-TIGIT and HT1080-aCD3scFv cell systems. Bar reaction

[0277] Experimental methods:

[0278] Jurkat cells were transfected with the TIGIT vector plenti6.3 / V5-TIGITFL-BSD (pLenti6.3 vector purchased from Invitrogen). Stable TIGIT-expressing cell lines, Jurkat-TIGIT cells, were obtained through selection. The anti-CD3 antibody vector pCDH-aCD3scFv-puro was prepared by GenScript (the cDNA sequence of anti-CD3scFv was optimized and cloned into the pUC57simple vector provided by GenScript, resulting in the pUC57simple-anti-CD3scFv plasmid). The pUC57simple-anti-CD3scFv plasmid was digested with XbaI and BamHI, and the anti-CD3scFv target gene fragment was recovered and subcloned into the pCDH-CMV-MCS-EF1-Puro expression vector (purchased from Eukaryotic Biotechnology) via the restriction sites XbaI and BamHI. (The anti-CD3scFv sequence is from the reference: Eukaryotic expression of anti-CD3 singlechain Fv antibody). gene and the characterization of its bioactivities JOURNALXi Bao Yu Fen Zi Mian Yi Xue Za Zhi 20(5),552-555(2004),PUBMED 15367345); Transfected HT-1080 cells, and after screening, the cell line HT1080-aCD3scFv cells that stably express anti-CD3scFv on the cell membrane were obtained.

[0279] Jurkat-TIGIT and HT1080-aCD3scFv cells in logarithmic growth phase were collected and added to each well of a 96-well plate. 50,000 cells were added to each well of Jurkat-TIGIT and 10,000 cells to each well of HT1080-aCD3scFV. Diluted antibodies (final concentrations of 10 nM, 50 nM, and 250 nM) and anti-human CD28 antibody (purchased from R&D Company, catalog number: MAB342-500) (3 μg / mL) were added, and the plates were incubated for 48 h. The culture supernatant was collected, and the IL-2 content was detected using an IL-2 ELISA kit.

[0280] Experimental results are as follows Figure 17 As shown.

[0281] The results showed that both the humanized antibody 26B12H2L2 and the positive control antibody RG6058 could promote the secretion of IL-2 in the system, and at each concentration (10nM, 50nM, 250nM), the level of IL-2 secretion promoted by humanized antibody 26B12H2L2 and RG6058 was comparable.

[0282] The results showed that the humanized antibody 26B12H2L2 had a comparable ability to induce cells to secrete IL-2 as the positive control antibody RG6058.

[0283] Example 10: Treatment of xenografts in hTigit-BALB / c transgenic mice inoculated with CT26 mice using 26B12H2L2 effect

[0284] A mouse tumor model was established by inoculating hTigit-BALB / c transgenic mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., where the normal TIGIT gene was replaced by the human TIGIT gene) with 500,000 CT26 cells (mouse colon cancer cell line, purchased from ATCC) in the back of each mouse. Specifically, 200 μl of CT26 cells at a density of 25 million / ml were injected into each mouse to establish the tumor model. Eight mice were used in each group: a control group (administered 20 mg / kg via intraperitoneal injection, twice weekly) and an experimental group (administered 20 mg / kg via intraperitoneal injection, twice weekly). The specific protocol is shown in Table 9.

[0285] Table 9: Modeling of the mouse CT26 tumor model and antibody dosing regimen

[0286]

[0287]

[0288] Experimental results are as follows Figure 18 As shown.

[0289] The results showed that 26B12H2L2 significantly reduced tumor volume in the hTIGIT-BALB / c transgenic mouse CT26 tumor model.

[0290] The results showed that 26B12H2L2 had strong efficacy in the hTIGIT-BALB / c transgenic mouse CT26 tumor model and has the potential for treatment and / or prevention of tumors, especially colorectal cancer.

[0291] At the same time, such as Figure 19 As shown, 26B12H2L2 had no effect on the body weight of hTIGIT-BALB / c transgenic mice, which served as a CT26 tumor model, indicating that the 26B12H2L2 antibody did not produce any toxic side effects in mice.

[0292] Example 11: Effective treatment with combination therapy of anti-TIGIT antibody and anti-CTLA4-anti-PD-1 bispecific antibody

[0293] To detect the in vivo tumor-suppressive activity of the anti-TIGIT antibody combined with the anti-CTLA4-anti-PD-1 bispecific antibody CP004 (hG1TM), CT26 cells (human colon cancer cells, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were first subcutaneously inoculated into 5-7 week old female BALB / c-hPD1 / hTIGIT mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). When the average tumor volume reached 80-120 mmHg, the tumor cells were inoculated into the target tumor cells. 3 Mice were randomly divided into 4 groups of 6 mice each, based on tumor volume. The day of grouping was defined as day D0, and drug administration began on day D0. In the combined drug administration group, the drugs were prepared separately and administered sequentially (there were no specific requirements for the order or interval of administration; one drug was administered before the next). Model establishment and specific administration methods are shown in Table 10. After drug administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0294] Table 10: Dosing regimen for the combined use of anti-TIGIT antibody and anti-CTLA4-anti-PD-1 bispecific antibody in the treatment of CT26 xenograft BALB / c-hPD1 / hTIGIT mouse model

[0295]

[0296] result Figure 20 As shown in the figure. The results showed that compared with the isotype control antibody hIgG, CP004(hG1TM) and 26B12H2L2 could effectively inhibit the growth of mouse tumors, and the CP004(hG1TM)+26B12H2L2 group showed combined anti-tumor efficacy in this model, and its combined inhibition of tumors was better than that of the test drug alone.

[0297] In addition, such as Figure 21 As shown, tumor-bearing mice tolerated the test drugs CP004 (hG1TM) and 26B12H2L2 well, both alone and in combination, and there was no effect on the body weight of tumor-bearing mice in any group.

[0298] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

[0299] Sequence List (Note: Underlined lines indicate CDR sequences)

[0300] The amino acid sequence of 26B12VH

[0301] EVQLQESGPGLVKPSQSLSLTCTVT GHSFTSDYA WNWIRQFPGNRLEWMGY ISYSDST NYNPSLKSRISITRDTSKNQFFLQMNSVTTEDTATYYC ARLDYGNYGGAMDY WGQ GTSVTVSS(SEQ ID NO: 1)

[0302] Nucleic acid sequence of 26B12VH

[0303] GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGAAACCCTCTCAGTCTCT GTCCCTCACCTGCACTGTCACT GGCCACTCATTCACCAGTGATTATGCC TGGAACTG GATCCGGCAGTTTCCAGGAAACAGACTGGAGTGGATGGGCTAC ATAAGCTACAGTGATAGCACT AACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACAT CCAAGAACCAGTTCTTCTTGCAGATGAATTCTGTGACTACTGAGGACACAGCCACA TATTACTGT GCAA GATTGGACTATGGTAACTACGGGTGGGGCTATGGACTAC TGGGG TCAAGGGACCTCAGTCACCGTCTCCTCA(SEQ ID NO: 2)

[0304] HCDR1: GHSFTSDYA(SEQ ID NO: 3)

[0305] HCDR2: ISYSDST(SEQ ID NO: 4)

[0306] HCDR3: ARLDYGNYGGAMDY(SEQ ID NO: 5)

[0307] Amino acid sequence of 26B12VL

[0308] DIVLTQSHEFMSTSLRDRVSITCKSS QHVSTA VAWYQQKPGQSPKLLIY SAS YRYT GVPDRFTGSGSGTDFTFTISSVKAEDLAVYYCQQHYITPWTFGGGTKLEIK(SEQ ID NO: 6)

[0309] Nucleic acid sequence of 26B12VL

[0310] GATATTGTGCTAACTCAGTCTCACGAATTCATGTCCACCTCATTACGAGACAGG GTCAGCATCACCTGCAAATCCAGT CAACATGTGAGTACTGCT GTAGCCTGGTATCA ACAGAAACCAGGACAATCTCCTAAACTACTGATTTAC TCGGCATCC TACCGGTACA CTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCA TCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGT CAGCAACATTATATTACTCCGTGGACG FGGGTKLDIK (SEQ ID NO: 7)

[0311] LCDR1: QHVSTA (SEQ ID NO: 8)

[0312] LCDR2: SAS (SEQ ID NO: 9)

[0313] LCDR3: QQHYITPWT (SEQ ID NO: 10)

[0314] Amino acid sequence of 26B12H1

[0315] DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WNWIRQFPGKGLEWIGY ISYSDST NYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 11)

[0316] Nucleic acid sequence of 26B12H1

[0317] GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCT GTCTCTGACCTGTACAGTGTCT GGCCACAGCTTCACATCCGACTACGCC TGGAACTG GATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATCGGCTAC ATCTCTTATAGCGACTCCACCAACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACA TCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCAC ATACTATTGC GCCC GGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC WGGG GCCAGGGCACCTCCGTGACAGTGAGCTCC(SEQ ID NO: 12)

[0318] Amino acid sequence of 26B12H2

[0319] DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WSWIRQPPGKGLEWIGY ISYSDST NYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYC ARLDYGNYGGAMDY WGQ GTSVTVSS(SEQ ID NO: 13)

[0320] Nucleic acid sequence of 26B12H2

[0321] GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCT GTCCCTGACCTGTACAGTGTCC GGCCACTCTTTTACAAGCGACTACGCC TGGTCTTG GATCAGGCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTAC ATCTCCTATTCTGACAGCACC AACTATAATCCCTCCCTGAAGTCTCGGGTGACCATCTCTAGAGATACA AGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGT GTACTATTGC GCCC GGCTGGACTACGGCAATTATGCGCGGAGCCATGGATTAC WGGG GCCAGGGCACCAGCGTGACAGTGTCTAGC(SEQ ID NO: 14)

[0322] Amino acid sequence of 26B12H3

[0323] DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WSWIRQPPGKGLEWIGY ISYSDST NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC ARLDYGNYGGAMDYWGQ GTSVTVSS(SEQ ID NO: 15)

[0324] Nucleic acid sequence of 26B12H3

[0325] GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCT GTCCCTGACCTGTACAGTGTCC GGCCACTCTTTTACAAGCGACTACGCC TGGTCTTG GATCAGACAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTAC ATCTCCTATTCTGACAGCACC AACTATAATCCCTCCCTGAAGTCTAGAGTGACCATCTCTGTGGATACA AGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGT GTACTATTGC GCCC GGCTGGACTACGGCAATTATGCGCGGAGCCATGGATTAC TGGG GCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 16)

[0326] Amino acid sequence of 26B12H4

[0327] DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WNWIRQFPGKGLEWMGY ISYSDST NYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYC ARLDYGNYGGAMDY WGQ GTSVTVSS(SEQ ID NO: 17)

[0328] Nucleic acid sequence of 26B12H4

[0329] GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCT GTCTCTGACCTGTACAGTGTCT GGCCACAGCTTCACATCCGACTACGCC TGGAACTG GATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATGGGCTAC ATCTCTTATAGCGACTCCACC AACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACA TCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCAC ATACTATTGC GCCC GGCTGGACTACGGCAATTATGCGCGGAGCCATGGATTACTGGG GCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 18)

[0330] Amino acid sequence of 26B12L1

[0331] DIQMTQSPKSLSTSVGDRVTITCRSS QHVSTA VAWYQQKPGKSPKLLIY SAS YRYS GVPDRFSGSGSGTDFTFTISSVQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 19)

[0332] Nucleic acid sequence of 26B12L1

[0333] GACATCCAGATGACCCAGTCCCCTAAGTCCCTGTCTACAAGCGTGGGCGATCG GGTGACCATCACATGTAGAAGCTCC CAGCACGTGTCTACCGCA GTGGCATGGTACC AGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAT TCCGCCTCT TACAGGTAT TCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACA ATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTACAT CACCCCATGGACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 20)

[0334] Amino acid sequence of 26B12L2

[0335] DIQMTQSPSSLSASVGDRVTITCRSS QHVSTA LAWYQQKPGKSPKLLIY SAS SRYSG VPDRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYITPWTFGGGTKLEIK (SEQ ID NO: 21)

[0336] Nucleic acid sequence of 26B12L2

[0337] GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCTGCCAGCGTGGGCGATAG GGTGACCATCACATGTAGATCTAGC CAGCACGTGTCTACAGCC Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr TCCGCCTCC Ser Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys CAGCAGCACTATATCA_CCCATGGACCT TCGGCGGCGGCACCAAGCTGGAGATCAAG( SEQ ID NO: 22)

[0338] Amino acid sequence of 26B12L3

[0339] DIQMTQSPSSLSASVGDRVTITCRAS QHVSTA LAWYQQKPGKAPKLLIY SAS SLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYITPWT FGGGTKLEIK(SEQ ID NO: 23)

[0340] Nucleic acid sequence of 26B12L3

[0341] GACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGATAG GGTGACCATCACATGTAGAGCCTCT CAGCACGTGAGCACAGCC Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr AGCGCCTCT Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys CAGCAGCACTACATC Thr Pro Trp Asp Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys(SEQ ID NO: 24)

[0342] Amino acid sequence of 26B12L4

[0343] DIQMTQSPKSMSTSVGDRVTITCRSS QHVSTA VAWYQQKPGKSPKLLIY SAS YRYSGVP DRFSGSGSGTDFTFTISSVQPEDFATYYC QQHYITPWT FGGGTKLEIK(SEQ ID NO:25)

[0344] Nucleic acid sequence of 26B12L4

[0345] GACATCCAGATGACCCAGTCCCCTAAGTCCATGTCTACAAGCGTGGGCGACAG GGTGACCATCACATGTAGAAGCTCC CAGCACGTGTCTACCGCA GTGGCATGGTACC AGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAT TCCGCCTCT TACAGGTAT TCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACA ATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACC TTCGGCGGCGGCACAAAGCTGGAGATCAAG(SEQ ID NO:26)

[0346] The following is the sequence of CTLA4-PD1.

[0347] The amino acid sequence of the 14C12 heavy chain variable region

[0348] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGG RYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGT LVTVSA(SEQ ID NO:.27)

[0349] The amino acid sequence of the 14C12 light chain variable region

[0350] DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVP SRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK(SEQ ID NO:.28)

[0351] HCDR1 of 14C12: Gly Phe Ala Phe Ser Ser Tyr Asp (SEQ ID NO: 29)

[0352] HCDR2 of 14C12: Ile Ser Gly Gly Gly Arg Tyr Thr (SEQ ID NO: 30)

[0353] HCDR3 of 14C12: Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr (SEQ ID NO: 31)

[0354] LCDR1 of 14C12:Gln Asp Ile Asn Thr Tyr (SEQ ID NO:32)

[0355] LCDR2 of 14C12:Arg Ala Asn(SEQ ID NO:33)

[0356] LCDR3 of 14C12:Leu Gln Tyr Asp Glu Phe Pro Leu Thr(SEQ ID NO:34)

[0357] The amino acid sequence of the 4G10 heavy chain variable region

[0358] QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYN NITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVT VSAAKTTPPSVY(SEQ ID NO:.35)

[0359] The amino acid sequence of the variable region of the 4G10 light chain

[0360] QAVVTQESALTSPGETVTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNR APGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSS PSVTLFQGQFC(SEQ ID NO:.36)

[0361] HCDR1 of 4G10: Gly Tyr Ser Phe Thr Gly Tyr Thr (SEQ ID NO:.37)

[0362] HCDR2 of 4G10:Ile Asn Pro Tyr Asn Asn Ile Thr(SEQ ID NO:.38)

[0363] HCDR3 of 4G10: Ala Arg Leu Asp Tyr Arg Ser Tyr (SEQ ID NO:.39)

[0364] LCDR1 of 4G10: Thr Gly Ala Val Thr Thr Ser Asn Phe (SEQ ID NO:.40)

[0365] LCDR2 of 4G10:Gly Thr Asn(SEQ ID NO:.41)

[0366] LCDR3 of 4G10: Ala Leu Trp Tyr Ser Asn His Trp Val (SEQ ID NO:42)

[0367] The amino acid sequence of the variable region of the 14C12H1L1 heavy chain

[0368] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGG GRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQ GTLVTVSS(SEQ ID NO:43)

[0369] The amino acid sequence of the variable region of the 14C12H1L1 light chain

[0370] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVP SRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK(SEQ ID NO:44)

[0371] The amino acid sequence of the variable region of the 4G10H1L1 heavy chain

[0372] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPY NNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGTLVT VSA(SEQ ID NO:45)

[0373] The amino acid sequence of the variable region of the 4G10H3L3 heavy chain

[0374] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPY NNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTLVT VSA(SEQ ID NO:46)

[0375] The amino acid sequence of 4G10H1V(M)

[0376] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQAPGQCLEWIGLINP YNNITNYNQKFMGKATFTVDKSISTAYMELSRLTSDDSGVYFCARLDYRSYWGQGT LVTVSA(SEQ ID NO:47)

[0377] The amino acid sequence of 4G10H3V(M)

[0378] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQCLEWIGLINP YNNITNYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFCARLDYRSYWGQGTL VTVSA(SEQ ID NO:48)

[0379] The amino acid sequence of the variable region of the 4G10H1L1 light chain

[0380] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTNN RASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL (SEQ ID NO:49)

[0381] The amino acid sequence of the variable region of the 4G10H3L3 light chain

[0382] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTNN KASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL (SEQ ID NO:50)

[0383] The amino acid sequence of 4G10L1V(M)

[0384] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQEKPGQAFRSLIGGTN NRASWVPARFSGSLLGGKAALTISGAQPEDEAEYFCALWYSNHWVFGCGTKLTVLR (SEQ ID NO:51)

[0385] The amino acid sequence of 4G10L3V(M)

[0386] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQKPGQAPRSLIGGTN NKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYCALWYSNHWVFGCGTKLTVLR (SEQ ID NO:52)

[0387] Amino acid sequence of the heavy chain of the immunoglobulin moiety in CP004(hG1TM)

[0388] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISG GGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:53)

[0389] The amino acid sequence encoded by the 14C12H1L1 light chain (14C12L1) is: (214aa)

[0390] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRL VSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 54)

[0391] Amino acid sequence of Linker 1: GGGGSGGGGSGGGGS(SEQ ID NO:55)

[0392] Amino acid sequence of Linker 2: GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:56)

[0393] Amino acid sequence of the heavy chain of CP004(hG1TM)

[0394] EVQLVESGGGLVQPGGSLRLSCAAS GFAFSSYD MSWVRQAPGKGLDWVAT ISGGGRYT YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC ANRYGEAWFAY WGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQVQLVESGAEVKKPGASVKVSCKAS GYSFTGYT MNWVRQAPGQCLEWIGL IN PYNNIT NYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFC ARLDYRSY WGQGTLVT VSAGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSS TGAVTTSNF P NWVQQKPGQAPRSLIG GTN NKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYC ALWYSNHWV FGCGTKLTVLR(SEQ ID NO:57)

[0395] Nucleic acid sequence of CP004(hG1TM) heavy chain

[0396] GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACT GCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGG AGATACACCTACTATCCTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCAC TGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGTGCCAGCACCAAAGGGCCCAGCGTGTTTCCTCTCGCC CCCTCCTCCAAAAGCACCAGCGGAGGAACCGCTGCTCTCGGATGTCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGAGCTGGAATAGCGGCGCTCTGACAAGCGGAG TCCATACATTCCCTGCTGTGCTGCAAAGCAGCGGACTCTATTCCCTGTCCAGCGTCGTCACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTCAACCAC AAGCCCTCCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAATCCTGCGACAAGACACACACCTGTCCCCCCTGTCCTGCTCCCGAAGCTGCTGGAGCCCCTAGCGTCTTCC TCTTTCCTCCCAAACCCAAGGACACCCTCATGATCAGCAGAACCCCTGAAGTCACCTGTGTCGTCGTGGATGTCAGCCATGAGGACCCCGAGGTGAAATTCAACTGGTATGTCGATGGCGTCGAGGTGCACAACGCCAAAACCAAGCCCAGGGAGGAACAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACAGTCCTCCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTCCCTGCCCCCATTGAGAAGAC CATCAGCAAGGCCAAAGGCCAACCCAGGGAGCCCCAGGTCTATACACTGCCTCCCTCCAGGGACGAACTCACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTT TATCCCAGCGACATCGCCGTCGAGTGGGAGTCCAACGGACAGCCCGAGAATAACTACAAGACCACCCCTCCTGTCCTCGACTCCGACGGCTCCTTCTTCCTGTACAGCAAACT GACCGTGGATAAGTCCAGATGGCAGCAGGGCAATGTCTTTTCATGTTCCGTGATGCACGAGGCACTGCACAACCACTATACCCAGAAGTCTCTGAGTCTGTCACCAGGAAAA GGAGGAGGAGGCTCTGGAGGAGGCGGAAGTGGAGGCGGAGGATCAGGAGGGGGA GGATCTCAGGTGCAGCTGGTCGAATCCGGAGCCGAGGTGAAGAAACCCGGCGCTTCCGTGAAGGTCTCTTGCAAAGCATCAGGCTACAGCTTCACAGGGTATACTATGAACT GGGTGCGGCAGGCACCTGGACAGTGTCTGGAATGGATCGGCCTGATTAACCCATACAACAACATCACTAACTACGCCCAGAAGTTCCAGGGCCGGGTGACTTTTACCGTGGA CACTAGCATTTCCACCGCTTACATGGAGCTGAGTCGGCTGAGATCAGACGATACCGGCGTGTATTTTTGCGCAAGGCTGGATTACAGAAGTTATTGGGGACAGGGAACACTTGTTACAGTCTCTGCTGGAGGAGGCGGATCTGGAGGAGGAGGATCTGGCGGAGGAGGCAGTGGAGGAGGAGGATCACAGGCTGTGGTTACTCAGGAACCAAGCCTGACCGT GAGCCCCGGAGGCACAGTCACTCTGACCTGTGGGAGCTCCACAGGAGCTGTGACCA CATCTAACTTCCCTAATTGGGTGCAGCAGAAGCCAGGACAGGCACCTCGATCCCTGATCGGGGGAACCAACAACAAGGCCAGCTGGACACCCGCCAGATTTTCTGGCAGTCT GCTGGGCGGGAAAGCCGCTCTGACCATTAGCGGCGCTCAGCCTGAGGACGAAGCAGAGTACTATTGCGCCCTGTGGTATAGTAATCATTGGGTGTTCGGGTGTGGGACAAA ACTGACCGTGCTGAGA(SEQ ID NO: 58)

[0397] Amino acid sequence of CP004(hG1TM) light chain

[0398] DIQMTQSPSSMSASVGDRVTFTCRAS QDINTY LSWFQQKPGKSPKTLIY RAN RLVS GVPSRFSGSGSGQDYTLTISSLQPEDMATYYC LQYDEFPLT FGAGTKLELKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 59)

[0399] Nucleic acid sequence of CP004(hG1TM) light chain

[0400] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTC ACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGG AGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCA GCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCC TCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTG GTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAA GATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAA GTCTTTTAACCGGGGCGAATGT(SEQ ID NO: 60)

[0401] Heavy chain CDR amino acid sequences of CP004(hG1TM)

[0402] HCDR1: GFAFSSYD(SEQ ID NO: 61)

[0403] HCDR2: ISGGGRYT(SEQ ID NO: 62)

[0404] HCDR3: ANRYGEAWFAY(SEQ ID NO: 63)

[0405] HCDR4: GYSFTGYT(SEQ ID NO: 64)

[0406] HCDR5: INPYNNIT(SEQ ID NO: 65)

[0407] HCDR6:ARLDYRSY (SEQ ID NO:66)

[0408] HCDR7:TGAVTTSNF(SEQ ID NO:67)

[0409] HCDR8:GTN (SEQ ID NO:68)

[0410] HCDR9:ALWYSNHWV (SEQ ID NO:69)

[0411] CP004(hG1TM) light chain CDR amino acid sequence

[0412] LCDR1:QDINTY(SEQ ID NO:70)

[0413] LCDR2:RAN(SEQ ID NO:71)

[0414] LCDR3:LQYDEFPLT(SEQ ID NO:72)

[0415] CP004(hG1TM) heavy chain variable region amino acid sequence

[0416] VH1:EVQLVESGGGLVQPGGSLRLSCAAS GFAFSSYD MSWVRQAPGKGLDWVAT ISGGGRYT YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC ANRYGEAWFAY W GQGTLVTVSS(SEQ ID NO:73)

[0417] VH2:QVQLVESGAEVKKPGASVKVSCKAS GYSFTGYT MNWVRQAPGQCLEWIGL INPYNNIT NYAQKFQGRVTFTVDTSISTAYMELSRLRSDDTGVYFC ARLDYRSY WGQGT LVTVSA (SEQ ID NO:74)

[0418] VH3:QAVVTQEPSLTVSPGGTVTLTCGSS TGAVTTSNF PNWVQQKPGQAPRSLIG GTN NKASWTPARFSGSLLGGKAALTISGAQPEDEAEYYC ALWYSNHWV FGCGTKLTVL R(SEQ ID NO:75)

[0419] Amino acid sequence of the light chain variable region of CP004(hG1TM)

[0420] DIQMTQSPSSMSASVGDRVTFTCRAS QDINTY LSWFQQKPGKSPKTLIY RAN RLVS GVPSRFSGSGSGQDYTLTISSLQPEDMATYYC LQYDEFPLT FGAGTKLELK(SEQ ID NO:76)

[0421] Amino acid sequence of mFc:

[0422] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERT ISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:77)

[0423] Sequence of hFc

[0424] THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78)

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. The anti-TIGIT antibody comprises HCDR1-HCDR3 and LCDR1-LCDR3, wherein HCDR1-HCDR3 are HCDR1-HCDR3 in the heavy chain variable region of SEQ ID NO:1 as determined according to the Kabat, IMGT, Chothia, or AbM numbering system, and LCDR1-LCDR3 are LCDR1-LCDR3 in the light chain variable region of SEQ ID NO:6 as determined according to the Kabat, IMGT, Chothia, or AbM numbering system. The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of 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; the anti-CTLA4-anti-PD-1 bispecific antibody has an Ig-scFv structure, wherein the immunoglobulin is IgG, and there are two single-chain antibodies, one end of each single-chain antibody being connected to the C-terminus of the two heavy chains of the immunoglobulin; in, According to the Kabat, IMGT, Chothia, or AbM numbering system, the immunoglobulin comprising HCDR1-HCDR3 and LCDR1-LCDR3, wherein HCDR1 is HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:27, and LCDR1-LCDR3 is LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:28; and the single-chain antibody comprising HCDR1-HCDR3 and LCDR1-LCDR3, wherein HCDR1-HCDR3 is HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:35, and LCDR1-LCDR3 is LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:36; or, According to the Kabat, IMGT, Chothia, or AbM numbering system, the immunoglobulin comprising HCDR1-HCDR3 and LCDR1-LCDR3, wherein HCDR1-HCDR3 is HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:35 and LCDR1-LCDR3 is LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:36; and the single-chain antibody comprising HCDR1-HCDR3 and LCDR1-LCDR3, wherein HCDR1-HCDR3 is HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:27 and LCDR1-LCDR3 is LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:

28. The antigen-binding fragments are selected from Fab, Fab', F(ab')2, Fv, humanized antibodies, chimeric antibodies, or biantibodies.

2. The pharmaceutical composition or kit of claim 1, wherein, according to the IMGT numbering system, the anti-TIGIT antibody comprises HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs: 3-5 and LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs: 8-10, respectively. Or the immunoglobulins described herein comprise HCDR1-HCDR3 as shown in SEQ ID NOs: 29-31 and LCDR1-LCDR3 as shown in SEQ ID NOs: 32-34, respectively; and the single-chain antibodies described herein comprise HCDR1-HCDR3 as shown in SEQ ID NOs: 37-39 and LCDR1-LCDR3 as shown in SEQ ID NOs: 40-42, respectively; or, The immunoglobulins comprise HCDR1-HCDR3 as shown in SEQ ID NOs: 37-39 and LCDR1-LCDR3 as shown in SEQ ID NOs: 40-42, respectively; and the single-chain antibodies comprise HCDR1-HCDR3 as shown in SEQ ID NOs: 29-31 and LCDR1-LCDR3 as shown in SEQ ID NOs: 32-34, respectively.

3. The pharmaceutical composition or kit according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is selected from SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or 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 or SEQ ID NO:

25.

4. The pharmaceutical composition or kit of claim 1 or 2, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

6. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

19. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

19. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

23. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

23. The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 25; or The amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:

25.

5. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The anti-TIGIT antibody includes a non-CDR region, and the non-CDR region is derived from a species that is not rodent.

6. The pharmaceutical composition or kit of claim 1 or 2, wherein the anti-TIGIT antibody comprises a non-CDR region, and the non-CDR region is derived from a human antibody.

7. The pharmaceutical composition or kit according to claim 1 or 2, wherein, The heavy chain constant region of the anti-TIGIT antibody is the Ig gamma-1 chain C region; the light chain constant region is the Ig kappa chain C region.

8. The pharmaceutical composition or kit according to claim 1 or 2, wherein, The heavy chain constant region of the anti-TIGIT antibody is shown in NCBI ACCESSION: P01857; the light chain constant region is shown in NCBI ACCESSION: P01834.

9. The pharmaceutical composition or kit of claim 1, wherein the antigen-binding fragment of the anti-TIGIT antibody is a single-chain antibody.

10. The pharmaceutical composition or kit according to claim 1 or 2, wherein, The anti-TIGIT antibody has a K value of less than 4E-10. D Combined with TIGIT-mFc.

11. The pharmaceutical composition or kit according to claim 1 or 2, wherein, The anti-TIGIT antibody has a K value of less than 4E-11. D Combined with TIGIT-mFc.

12. The pharmaceutical composition or kit of claim 10 or 11, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The K D Measured using a Fortebio molecular interaction analyzer.

13. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The anti-TIGIT antibody was expressed at an EC50 concentration of less than 1.5 nM. 50 Combined with TIGIT-mFc.

14. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The anti-TIGIT antibody has an EC50 of less than 1.2 nM. 50 Combined with TIGIT-mFc.

15. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The anti-TIGIT antibody was expressed at an EC50 concentration of less than 1 nM. 50 Combined with TIGIT-mFc.

16. The pharmaceutical composition or kit according to any one of claims 13-15, wherein the anti-TIGIT antibody or its antigen-binding fragment, wherein, The EC 50 Measured by flow cytometry.

17. The pharmaceutical composition or kit of claim 1 or 2, wherein the anti-TIGIT antibody is a monoclonal antibody, a humanized antibody, or a chimeric antibody.

18. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-TIGIT antibody is an antibody produced by the hybridoma cell line LT019, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2020208.

19. The pharmaceutical composition or kit according to claim 1 or 2, wherein the 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 NO: 27, SEQ ID NO: 43 or a sequence having at least 89% homology thereto; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 28, SEQ ID NO: 44 or a sequence having at least 90% 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 or a sequence having at least 80% homology therewith, SEQ ID NO: 45 or a sequence having at least 89% homology therewith, SEQ ID NO: 46 or a sequence having at least 80% homology therewith, SEQ ID NO: 47 or a sequence having at least 89% homology therewith, and SEQ ID NO: 48 or a sequence having at least 80% homology therewith; and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 36 or a sequence having at least 80% homology therewith, SEQ ID NO: 49 or a sequence having at least 85% homology therewith, SEQ ID NO: 50 or a sequence having at least 80% homology therewith, SEQ ID NO: 51 or a sequence having at least 84% homology therewith, and SEQ ID NO: 52 or a sequence having at least 80% homology therewith. or, The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 35 or a sequence having at least 80% homology therewith, SEQ ID NO: 45 or a sequence having at least 89% homology therewith, SEQ ID NO: 46 or a sequence having at least 80% homology therewith, SEQ ID NO: 47 or a sequence having at least 89% homology therewith, and SEQ ID NO: 48 or a sequence having at least 80% homology therewith; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 36 or a sequence having at least 80% homology therewith, SEQ ID NO: 49 or a sequence having at least 85% homology therewith, SEQ ID NO: 50 or a sequence having at least 80% homology therewith, SEQ ID NO: 51 or a sequence having at least 84% homology therewith, and SEQ ID NO: 52 or a sequence having at least 80% homology therewith; 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 89% homology thereto; 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 90% homology thereto.

20. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, The bispecific antibody is selected from any one of (1)-(20) below: (1) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 28 or a sequence having at least 90% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto; (2) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 28 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 45 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 49 or a sequence having at least 85% homology thereto; (3) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:28 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:46 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:50 or a sequence having at least 80% homology thereto; (4) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto; (5) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 45 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 49 or a sequence having at least 85% homology thereto; (6) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto; (7) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:35 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:36 or a sequence having at least 80% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:28 or a sequence having at least 89% homology thereto; (8) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 35 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 36 or a sequence having at least 80% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; (9) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO:45 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO:49 or a sequence having at least 85% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO:27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO:28 or a sequence having at least 89% homology thereto; (10) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 45 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 49 or a sequence having at least 85% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; (11) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 28 or a sequence having at least 89% homology thereto; (12) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 46 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 50 or a sequence having at least 80% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; (13) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 28 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 47 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 51 or a sequence having at least 84% homology thereto; (14) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 28 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto; (15) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 47 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 51 or a sequence having at least 84% homology thereto; (16) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto; (17) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 47 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 51 or a sequence having at least 84% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 28 or a sequence having at least 89% homology thereto; (18) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 27 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 28 or a sequence having at least 89% homology thereto; (19) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 47 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 51 or a sequence having at least 84% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto; as well as, (20) The amino acid sequence of the heavy chain variable region of the immunoglobulin is as shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the immunoglobulin is as shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto; and the amino acid sequence of the heavy chain variable region of the single-chain antibody is as shown in SEQ ID NO: 43 or a sequence having at least 89% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is as shown in SEQ ID NO: 44 or a sequence having at least 89% homology thereto.

21. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 91% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 91% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 81% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 81% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 90% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 81% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 90% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 81% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 86% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 81% homology with SEQ ID NO:50; The homologous sequence of SEQ ID NO:51 has at least 85% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 81% homology with SEQ ID NO:

52.

22. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 92% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 92% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 82% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 82% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 91% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 82% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 91% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 82% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 87% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 82% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 86% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 82% homology with SEQ ID NO:

52.

23. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 93% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 93% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 83% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 83% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 92% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 83% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 92% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 83% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 88% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 83% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 87% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 83% homology with SEQ ID NO:

52.

24. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 94% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 94% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 84% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 84% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 93% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 84% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 93% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 84% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 89% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 84% homology with SEQ ID NO:50; The homologous sequence of SEQ ID NO:51 has at least 88% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 84% homology with SEQ ID NO:

52.

25. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 95% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 95% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 85% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 85% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 94% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 85% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 94% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 85% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 90% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 85% homology with SEQ ID NO:50; The homologous sequence of SEQ ID NO:51 has at least 89% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 85% homology with SEQ ID NO:

52.

26. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 96% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 96% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 86% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 86% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 95% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 86% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 95% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 86% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 91% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 86% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 90% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 86% homology with SEQ ID NO:

52.

27. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 97% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 97% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 87% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 87% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 96% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 87% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 96% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 87% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 92% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 87% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 91% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 87% homology with SEQ ID NO:

52.

28. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 98% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 98% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 88% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 88% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 97% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 88% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 97% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 88% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 93% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 88% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 92% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 88% homology with SEQ ID NO:

52.

29. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:27 has at least 99% homology with SEQ ID NO:27; The homology sequence of SEQ ID NO:28 has at least 99% homology with SEQ ID NO:28; The homology sequence of SEQ ID NO:35 has at least 89% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 89% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 98% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 89% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 98% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 89% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 94% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 89% homology with SEQ ID NO:50; The homologous sequence of SEQ ID NO:51 has at least 93% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 89% homology with SEQ ID NO:

52.

30. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 90% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 90% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:45 has at least 99% homology with SEQ ID NO:45; The homology sequence of SEQ ID NO:46 has at least 90% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:47 has at least 99% homology with SEQ ID NO:47; The homology sequence of SEQ ID NO:48 has at least 90% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 95% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 90% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 94% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 90% homology with SEQ ID NO:

52.

31. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 91% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 91% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 91% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 91% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 96% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 91% homology with SEQ ID NO:50; The homologous sequence of SEQ ID NO:51 has at least 95% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 91% homology with SEQ ID NO:

52.

32. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 92% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 92% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 92% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 92% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 97% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 92% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 96% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 92% homology with SEQ ID NO:

52.

33. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 93% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 93% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 93% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 93% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 98% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 93% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 97% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 93% homology with SEQ ID NO:

52.

34. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 94% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 94% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 94% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 94% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:49 has at least 99% homology with SEQ ID NO:49; The homology sequence of SEQ ID NO:50 has at least 94% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 98% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 94% homology with SEQ ID NO:

52.

35. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 95% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 95% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 95% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 95% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:50 has at least 95% homology with SEQ ID NO:50; The homology sequence of SEQ ID NO:51 has at least 99% homology with SEQ ID NO:51; or The homology sequence of SEQ ID NO:52 has at least 95% homology with SEQ ID NO:

52.

36. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 96% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 96% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 96% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 96% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:50 has at least 96% homology with SEQ ID NO:50; or The homology sequence of SEQ ID NO:52 has at least 96% homology with SEQ ID NO:

52.

37. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 97% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 97% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 97% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 97% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:50 has at least 97% homology with SEQ ID NO:50; or The homology sequence of SEQ ID NO:52 has at least 97% homology with SEQ ID NO:

52.

38. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 98% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 98% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 98% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 98% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:50 has at least 98% homology with SEQ ID NO:50; or The homology sequence of SEQ ID NO:52 has at least 98% homology with SEQ ID NO:

52.

39. The pharmaceutical composition or kit according to claim 19 or 20, wherein... The homology sequence of SEQ ID NO:35 has at least 99% homology with SEQ ID NO:35; The homology sequence of SEQ ID NO:36 has at least 99% homology with SEQ ID NO:36; The homology sequence of SEQ ID NO:46 has at least 99% homology with SEQ ID NO:46; The homology sequence of SEQ ID NO:48 has at least 99% homology with SEQ ID NO:48; The homology sequence of SEQ ID NO:50 has at least 99% homology with SEQ ID NO:50; or The homology sequence of SEQ ID NO:52 has at least 99% homology with SEQ ID NO:

52.

40. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 53, and the amino acid sequence of its light chain is shown in SEQ ID NO:

54.

41. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, The first protein functional region is directly connected to the second protein functional region or connected through a linker fragment; and / or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linker fragment.

42. The pharmaceutical composition or kit of claim 41, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, wherein, The connection segment is (GGGGS)n, where n is a positive integer.

43. The pharmaceutical composition or kit of claim 42, wherein n is 1, 2, 3, 4, 5 or 6.

44. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, The variable region of the heavy chain of the single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin.

45. The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, The immunoglobulin is human IgG1 subtype; According to the EU numbering system, the heavy chain constant region of the immunoglobulin has one of the following combinations of mutations: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, G237A.

46. ​​The pharmaceutical composition or kit according to claim 1 or 2, wherein the anti-CTLA4-anti-PD-1 bispecific antibody, The bispecific antibody includes: Targeting the first protein functional region of PD-1, and Targeting the second protein functional region of CTLA4; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:53, and the amino acid sequence of its light chain is shown in SEQ ID NO:

54. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 48 or a sequence having at least 80% homology thereto, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 52 or a sequence having at least 80% homology thereto. The single-chain antibody is attached to the C-terminus of the heavy chain of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker fragment; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker fragment; the first linker fragment and the second linker fragment may be the same or different.

47. The pharmaceutical composition or kit of claim 46, wherein the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NO: 55 and SEQ ID NO:

56.

48. The pharmaceutical composition or kit of claim 47, wherein the amino acid sequences of the first linker fragment and the second linker fragment are both as shown in SEQ ID NO:

56.

49. The pharmaceutical composition or kit of claim 46, wherein the heavy chain amino acid sequence of the anti-CTLA4-anti-PD-1 bispecific antibody is shown in SEQ ID NOs:57, the light chain amino acid sequence is shown in SEQ ID NOs:59, and the bispecific antibody structure is IgG-scFv, wherein the IgG portion is an anti-PD1 antibody, and the scFv portion is an anti-CTLA4 antibody. The HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:61, the HCDR2 sequence in SEQ ID NO:62, the HCDR3 sequence in SEQ ID NO:63, and the VH sequence in SEQ ID NO:

73. Similarly, the LCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO:70, the LCDR2 sequence in SEQ ID NO:71, the LCDR3 sequence in SEQ ID NO:72, and the VL sequence 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.

50. The pharmaceutical composition or kit of claim 1 or 2, wherein the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-CTLA4-anti-PD-1 antibody is (1:5) to (5:1) based on the mass of the antibody.

51. The pharmaceutical composition or kit of claim 1 or 2, wherein the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-CTLA4-anti-PD-1 antibody is 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1, calculated by the mass of the antibody.

52. The pharmaceutical composition or kit according to claim 1 or 2, wherein, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

53. The pharmaceutical composition or kit according to claim 1 or 2, wherein, The pharmaceutical composition also includes excipients.

54. A reagent kit comprising individually packaged first and second products, wherein, The first product comprises the anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 1-18; The second product comprises the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49; The kit also includes an individually packaged third product containing one or more chemotherapy drugs.

55. The kit of claim 54, wherein the first product and the second product further comprise one or more pharmaceutically acceptable excipients independently.

56. The kit of claim 54, wherein the kit further comprises a product instruction manual.

57. The kit according to any one of claims 54-56, wherein, Based on antibody mass calculation, the mass ratio of anti-TIGIT antibody or its antigen-binding fragment to anti-CTLA4-anti-PD-1 bispecific antibody is (1:5)-(5:1).

58. The kit according to any one of claims 54-56, wherein, Based on antibody mass calculations, the mass ratio of anti-TIGIT antibody or its antigen-binding fragment to anti-CTLA4-anti-PD-1 bispecific antibody is 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:

1.

59. The use of an effective amount of the anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 1-18 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49 in the preparation of a medicament or kit for treating and / or preventing tumors, wherein the tumor is selected from one or more of the following: breast cancer, ovarian cancer, cervical cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, mesothelioma, lung cancer, liver cancer, gastrointestinal cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, and bone cancer.

60. The use of an effective amount of the anti-TIGIT antibody or antigen-binding fragment thereof as defined in any one of claims 1-18 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49 in the preparation of a medicament or kit for treating and / or preventing tumors, wherein the tumor is adenocarcinoma.

61. The use of an effective amount of the anti-TIGIT antibody or antigen-binding fragment thereof as defined in any one of claims 1-18 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49 in the preparation of a medicament or kit for the treatment and / or prevention of tumors, wherein the tumor is selected from one or more of the following: highly microsatellite unstable (MSI-H) and mismatch repair deficient (dMMR) solid tumors.

62. The use of an effective amount of the anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 1-18 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49 in the preparation of a medicament or kit for treating and / or preventing tumors, wherein the tumor is selected from one or more of the following: esophageal squamous cell carcinoma, gastric cancer, intestinal cancer, melanoma, osteosarcoma, chondrosarcoma, pancreatic cancer, small cell lung cancer, hepatocellular carcinoma, hepatobiliary cancer, and cholangiocarcinoma.

63. The use of an effective amount of the anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 1-18 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49 in the preparation of a medicament or kit for treating and / or preventing tumors, wherein the tumor is colorectal cancer.

64. The use of an effective amount of the anti-TIGIT antibody or antigen-binding fragment thereof as defined in any one of claims 1-18 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49 in the preparation of a medicament or kit for treating and / or preventing tumors, wherein the tumor is colon cancer.

65. The application according to any one of claims 59-64, wherein the drug or kit further comprises one or more other drugs.

66. The application of claim 65, wherein the other drug is selected from chemotherapeutic agents, targeted therapeutic agents, antibiotics, anti-hormonal agents, and / or hormonal drugs.

67. The application of claim 65, wherein the other drug is a growth inhibitor.

68. The application of claim 65, wherein the other drug is a cytotoxic agent.

69. The application of claim 65, wherein the other drug is selected from antimetabolites, antitumor antibiotics, plant-based anticancer drugs, and / or antibody-drug conjugates.

70. The application according to claim 65, wherein, The other drugs are selected from one or more of the following: alkylating agents, anthracyclines, antiandrogens, aromatase inhibitors, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, topoisomerase inhibitors, proteasome inhibitors, antimicrotubule agents, rhodopsin inhibitors, and histone deacetylase inhibitors.

71. The application according to claim 65, wherein, The other drugs are selected from one or more of the following: doxorubicin, asparaginase, platinum drugs, fluorouracil antitumor drugs, cyclophosphamide, pemetrexed, paclitaxel, and vinca alkaloids.

72. The application according to claim 65, wherein, The other drugs are selected from one or more of the following: EGFR antagonists, B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, and bisphosphatidylinositol 3-kinase / mTOR inhibitors.

73. The application according to claim 65, wherein, The other drugs are selected from one or more of the following: maydencin, monomethyl auristatin E, galicolamycin, goserelin, tamoxifen, medroxyprogesterone acetate, esperamicin, and radioisotope chelators.

74. The application according to claim 65, wherein, The other drugs are selected from one or more of the following: cisplatin, carboplatin, oxaliplatin, and tyrosine kinase inhibitors.

75. The application according to any one of claims 59-65, wherein, The anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4-anti-PD-1 bispecific antibody, are in a form suitable for intravenous injection or intravenous infusion.

76. The application of claim 66, wherein the chemotherapeutic agent is in a form suitable for intravenous injection or intravenous infusion.

77. The application according to claim 75, wherein, The anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4-anti-PD-1 bispecific antibody, are in liquid form.

78. The application of claim 66, wherein the chemotherapeutic agent is in liquid form.

79. The application according to any one of claims 59-65, wherein, The single-dose dose of the anti-TIGIT antibody or its antigen-binding fragment and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 0.1-100 mg per kilogram of body weight.

80. The application according to any one of claims 59-65, wherein, The single-dose dose of the anti-TIGIT antibody and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 1-10 mg per kilogram of body weight.

81. The application according to any one of claims 59-65, wherein, The single-dose dose of the anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 10-1000 mg per subject.

82. The application according to any one of claims 59-65, wherein, The single dose of the anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 50-500 mg.

83. The application according to any one of claims 59-65, wherein, The single dose of the anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 100-400 mg.

84. The application according to any one of claims 59-65, wherein, The single dose of the anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 150-300 mg.

85. The application according to any one of claims 59-65, wherein, The single dose of the anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 150-250 mg.

86. The application according to any one of claims 59-65, wherein, The single dose of the anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody is 200 mg.

87. The application according to any one of claims 59-65, wherein, The anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 anti-PD-1 bispecific antibody, may be administered twice daily to every other day, or once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

88. The application according to any one of claims 59-65, wherein, The anti-TIGIT antibody or its antigen-binding fragment, and / or the anti-CTLA4 and anti-PD-1 bispecific antibody, are in a form suitable for intravenous infusion or intravenous injection.

89. Unit dosage form, of which, The unit formulation comprises: 1 to 10,000 mg of the anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 1-18 and 1 to 10,000 mg of the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 1 and 19-49.

90. The unit formulation of claim 89, wherein the unit formulation comprises: 10-1000 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 1-1000 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

91. The unit formulation of claim 89, wherein the unit formulation comprises: 50-500 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 50-500 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

92. The unit formulation of claim 89, wherein the unit formulation comprises: 100-400 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 100-400 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

93. The unit formulation of claim 89, wherein the unit formulation comprises: 150-300 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 150-300 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

94. The unit formulation of claim 89, wherein the unit formulation comprises: 150-250 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 150-250 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

95. The unit formulation of claim 89, wherein the unit formulation comprises: 200 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 200 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

96. The unit formulation of claim 89, wherein the unit formulation comprises: 100 mg of the anti-TIGIT antibody or its antigen-binding fragment, or 100 mg of the aforementioned anti-CTLA4-anti-PD-1 bispecific antibody.

97. The unit formulation of claim 89, wherein the unit formulation further comprises one or more chemotherapeutic agents as defined in claim 71; wherein, The anti-TIGIT antibody or its antigen-binding fragment, the anti-CTLA4-anti-PD-1 bispecific antibody, and the chemotherapy agent are each packaged separately.

98. The unit formulation of claim 97, wherein the chemotherapeutic agent is a platinum-based drug and / or a fluorouracil-based antitumor drug.

99. The unit formulation according to any one of claims 89-98, for the treatment of tumors.

100. A single drug dose unit comprising the anti-TIGIT antibody or its antigen-binding fragment as defined in any one of claims 89-96 and the anti-CTLA4-anti-PD-1 bispecific antibody as defined in any one of claims 89-96.

101. The single-dose drug unit of claim 100, used for treating tumors.

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