Human tigit antibodies and uses thereof

Abstract

The application belongs to the technical field of antibodies, and discloses a human TIGIT antibody and application thereof. The human TIGIT antibody or antigen binding fragment thereof has strong binding capacity with hTIGIT, can induce B-hTIGIT transgenic mouse T cells, Jurkat cells and human PBMC cells to secrete cytokines, has significant ADCC effect and CDC effect, can significantly reduce the tumor volume / weight of a colon cancer CT26 cell strain tumor-bearing mouse and a breast cancer cell strain 4T1 tumor-bearing mouse without affecting the body weight of the mouse, prolongs the survival time of the mouse, has good pharmacokinetic characteristics, and can be used for detecting the expression level of hTIGIT and treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to human TIGIT antibodies and their applications. Background Technology

[0002] hTIGIT is a newly discovered costimulatory molecule with immunosuppressive effects. Researchers sequenced activated human T cells and further investigated proteins with immunomodulatory-like domains. The results showed that both T cells and natural killer cells (NK cells) express hTIGIT. This molecule possesses an immunoglobulin-like domain, a transmembrane region, and an immunoreceptor protein tyrosine inhibitory motif (ITIM), hence its name hTIGIT (T cell immunoglobulin and ITIM domain). The hTIGIT gene is located on human chromosome 16 and encodes a type I transmembrane protein composed of 244 amino acids. Its sequence is relatively conserved, and homologous molecules have been found in various mammals. The human hTIGIT molecule shares 88%, 67%, and 58% homology with the hTIGIT molecules in monkeys, dogs, and mice, respectively.

[0003] Among the ligands of hTIGIT, bioinformatics analysis and in vitro experiments revealed that hTIGIT has the highest affinity for CD155, followed by CD113, and a lower affinity for CD112. Studies have shown that after hTIGIT is activated by these ligands, it can inhibit T cell activation by downregulating three important upstream molecules in the TCR activation pathway: TCRα, CD3ε, and PLC-γ1. However, it also upregulates molecules that maintain T cell survival, such as IL-2Rγ, CD25, and BCL-XL, thus preventing T cells from being eliminated. NK cells are important immune cells in the body, playing a crucial role in anti-tumor, antiviral, and intracellular parasitic activity. Stanietsky et al. found that hTIGIT is not only expressed on the surface of T cells but also at high levels on the surface of NK cells. It can transmit inhibitory signals through its intracellular ITIM motif, inhibiting the killing function of NK cells, thereby exerting a direct inhibitory effect on NK cells. Figure 27 ).

[0004] Previous studies have found that hTIGIT expression on the surface of T cells is elevated during tumor progression, and that NK cells within the tumor express more hTIGIT compared to those surrounding the tumor. In the tumor immune cycle, hTIGIT can block the killing of tumor cells by immune cells through multiple steps. First, hTIGIT can inhibit NK cell effects by preventing the initial death of tumor cells and the release of tumor antigens. hTIGIT can also inhibit the co-stimulatory capacity of dendritic cells, leading to a decrease in cancer antigen presentation and an increase in anti-inflammatory cytokines such as IL-10, and can induce PVR signaling in other cells, such as tumor cells. Additionally, hTIGIT can inhibit CD8+ T cell effectors or skew CD4+ T cell polarization. Finally, hTIGIT can directly inhibit CD8+ T cell effectors, preventing the clearance of cancer cells. Figure 27 ).

[0005] It is evident that hTIGIT has a significant inhibitory effect on immune cells killing tumors; therefore, inhibiting this molecule may become an effective anti-tumor target. Studies have shown that a high-affinity, blocking monoclonal antibody targeting mouse hTIGIT has demonstrated a potent blocking effect on hTIGIT ligand binding and an enhancement effect on NK cell function in in vitro experiments. Furthermore, it has been demonstrated that hTIGIT-based checkpoint immunotherapy can reverse NK cell depletion, enhance NK cell-mediated anti-tumor immune responses, effectively inhibit tumor growth in mice, and significantly prolong the survival of tumor-bearing mice. In addition, this study also confirmed that NK cells are a prerequisite for the efficacy of other checkpoint immunotherapies (such as anti-PD-L1). Mice successfully treated with this hTIGIT monoclonal antibody possess near-lifetime potent anti-tumor immune memory and exhibit strong resistance to re-emergence of tumors without any further treatment. In conclusion, monoclonal antibodies targeting the inhibitory receptor hTIGIT have enormous drug development potential and show great promise for the treatment of solid tumors.

[0006] As of August 2021, according to information retrieved from the Biotech Gate database, clinical research targeting TIGIT is actively progressing. In China, BeiGene's BMS-986207 is the most advanced, while internationally, Roche's Tiragolumab (4.1D4, the reference antibody in this application) has entered Phase 3 clinical trials. All of these studies are for solid tumors. Table 1 shows the clinical trial status of various pharmaceutical companies targeting TIGIT.

[0007] Table 1. Summary of key clinical studies on human TIGIT targets (as of August 2021)

[0008] Summary of the Invention

[0009] The first aspect of the present invention aims to provide a human TIGIT antibody or an antigen-binding fragment thereof.

[0010] A second aspect of the present invention aims to provide a nucleic acid molecule encoding a human TIGIT antibody or an antigen-binding fragment thereof of the first aspect of the present invention.

[0011] A third aspect of this invention aims to provide an expression cassette, recombinant vector, or transgenic cell line comprising the nucleic acid molecule of the second aspect of this invention. A fourth aspect of this invention aims to provide an immunoconjugate.

[0012] The fifth aspect of this invention aims to provide the use of the human TIGIT antibody or antigen-binding fragment thereof of the first aspect of this invention, the nucleic acid molecule of the second aspect of this invention, the expression cassette of the third aspect of this invention, the recombinant vector or transgenic cell line and / or the immunoconjugate of the fourth aspect of this invention.

[0013] The sixth aspect of this invention aims to provide a product.

[0014] A seventh aspect of the present invention is to provide a pharmaceutical composition.

[0015] The object of the eighth aspect of the present invention is to provide a method for treating tumors.

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0017] In a first aspect, the present invention provides a human TIGIT antibody or an antigen-binding fragment thereof, which is 60H5 or 51C1;

[0018] The 60H5 includes a 60H5 heavy chain variable region and a 60H5 light chain variable region;

[0019] The 60H5 heavy chain variable region includes CDR1, CDR2, and CDR3;

[0020] The amino acid sequence of the CDR1 variable region of the 60H5 heavy chain is as follows:

[0021] a) GYTFTEYT (SEQ ID NO.2); or

[0022] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.2;

[0023] The amino acid sequence of the CDR2 variable region of the 60H5 heavy chain is as follows:

[0024] a) INPNGGT (SEQ ID NO.3); or

[0025] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.3;

[0026] The amino acid sequence of the CDR3 variable region of the 60H5 heavy chain is as follows:

[0027] a)ARSGNWDYAMDY(SEQ ID NO.4); or

[0028] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.4;

[0029] The variable region of the 60H5 light chain includes CDR1, CDR2, and CDR3;

[0030] The amino acid sequence of the CDR1 variable region of the 60H5 light chain is as follows:

[0031] a) QHVSTA (SEQ ID NO.7); or

[0032] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 7;

[0033] The amino acid sequence of the CDR2 variable region of the 60H5 light chain is as follows:

[0034] a) SAS (SEQ ID NO. 8); or

[0035] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 8;

[0036] The amino acid sequence of the CDR3 variable region of the 60H5 light chain is as follows:

[0037] a) QQHYITPWT (SEQ ID NO.9); or

[0038] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 9;

[0039] The 51C1 includes a 51C1 heavy chain variable region and a 51C1 light chain variable region;

[0040] The 51C1 heavy chain variable region includes CDR1, CDR2, and CDR3;

[0041] The amino acid sequence of the 51C1 heavy chain variable region CDR1 is as follows:

[0042] a)GYTFTEYF(SEQ ID NO.12); or

[0043] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 12;

[0044] The amino acid sequence of the 51C1 heavy chain variable region CDR2 is as follows:

[0045] a) FYPGSGSI (SEQ ID NO.13); or

[0046] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 13;

[0047] The amino acid sequence of the 51C1 heavy chain variable region CDR3 is as follows:

[0048] a)ARHEMRYGNYVLDY(SEQ ID NO.14); or

[0049] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 14;

[0050] The variable region of the 51C1 light chain includes CDR1, CDR2, and CDR3;

[0051] The amino acid sequence of the 51C1 light chain variable region CDR1 is as follows:

[0052] a) TGAVTTRNY (SEQ ID NO.17); or

[0053] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 17;

[0054] The amino acid sequence of the 51C1 light chain variable region CDR2 is as follows:

[0055] a) GTN (SEQ ID NO.18); or

[0056] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 18;

[0057] The amino acid sequence of the 51C1 light chain variable region CDR3 is as follows:

[0058] a) GLWYSNHLV (SEQ ID NO.19); or

[0059] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 19.

[0060] Preferably, the amino acid sequence of the 60H5 heavy chain variable region is as follows:

[0061] a)EVQLQQSGPELVKPGASLKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNNGGTKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARSGNWDYAMDYWGQGTSVTVSS(SEQ ID NO.1); or

[0062] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 1; or

[0063] c) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGQRLEWIGGINPNNGGTSYNQKFQGRVTITVDTSASTAYMELSSLRSEDTAVYYCARSGNWDYAMDYWGQGTTVTVSS (SEQ ID NO. 21); or

[0064] d) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 21;

[0065] The amino acid sequence of the variable region of the 60H5 light chain is as follows:

[0066] a)DIVMTQSHKFMSTSVGDRVSITCKASQHVSTAVVWYQQKPGQSPKLLIYSASYRYTGVDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYITPWTFGGGTKLEIKRADA(SEQ ID NO.6); or

[0067] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 6; or

[0068] c) DIQMTQSPSSMSASVGDRVTITCKASQHVSTAVVWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYITPWTFGGGTKLEIKRTVA (SEQ ID NO. 22); or

[0069] d) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 22;

[0070] The amino acid sequence of the 51C1 heavy chain variable region is as follows:

[0071] a) QVQLQQSGAELVKPGASVKLSCKASGYTFTEYFIHWIKQKSGQGLEWIGWFYPGSGSIKYNERFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHEMRYGNYVLDYWGQGTTLTVSS (SEQ ID NO.11); or

[0072] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 11; or

[0073] c) QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYFIHWVRQAPGQGLEWIGWFYPGSGSIKYNERFKDRVTLTADTSISTAYMELSRLRSDDTAVYYCARHEMRYGNYVLDYWGQGTTVTVSS (SEQ ID NO. 23); or

[0074] d) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 23;

[0075] The amino acid sequence of the 51C1 light chain variable region is as follows:

[0076] a) QAVVTQESALTITSPGETVTTLTCRSSTGAVTTRNYANWVQEKPDHLFTGLIGGTNNRVPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCGLWYSNHLVFGGGTKLTVLGQPK (SEQ ID NO.16); or

[0077] b) A sequence having 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO. 16; or

[0078] c) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTRNYANWVQQKPGQAPRGLIGGTNNRVPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCGLWYSNHLVFGGGTKLTVLGQPKA (SEQ ID NO. 24); or

[0079] d) A sequence having 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.24.

[0080] A second aspect of the invention provides a nucleic acid molecule encoding a human TIGIT antibody or an antigen-binding fragment thereof of the first aspect of the invention.

[0081] A third aspect of the invention provides an expression cassette, recombinant vector, or transgenic cell line comprising the nucleic acid molecule of the second aspect of the invention.

[0082] Preferably, the transgenic cell line does not contain any plant or animal species.

[0083] A fourth aspect of the present invention provides an immunoconjugate comprising a human TIGIT antibody or its antigen-binding fragment monoclonal antibody or its antigen-binding fragment, as described in the first aspect of the present invention, and a conjugation portion.

[0084] The conjugated portion is at least one of the following: a detectable marker, a drug, a toxin, a cytokine, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radionuclide, an enzyme, gold nanoparticles / nanorobars, magnetic nanoparticles, and a viral capsid protein.

[0085] Preferably, the detectable marker is a fluorescent or luminescent marker.

[0086] Preferably, the radionuclide is at least one of diagnostic isotopes and therapeutic isotopes.

[0087] Preferably, the diagnostic isotope is at least one selected from Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, and Re-188.

[0088] Preferably, the therapeutic isotope is at least one selected from Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, and Yb-177.

[0089] Preferably, the drug is a cytotoxic drug.

[0090] Preferably, the cytotoxic drug is at least one of the following: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, and vinca alkaloids; further, it is auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or drugs containing benzodiazepines. Drugs (such as at least one of pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, oxazolidinobenzodiazepines, and vincaalkaloids).

[0091] The fifth aspect of the present invention provides the use of the human TIGIT antibody or antigen-binding fragment thereof of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression cassette, recombinant vector or transgenic cell line of the third aspect of the present invention, and the immunoconjugate of the fourth aspect of the present invention.

[0092] Application of any one of (1) to (4) in any one of (5) to (6);

[0093] (1) The human TIGIT antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0094] (2) The nucleic acid molecule of the second aspect of the present invention;

[0095] (3) The expression cassette, recombinant vector, or transgenic cell line of the third aspect of the present invention;

[0096] (4) The immunoconjugate of the fourth aspect of the present invention;

[0097] (5) Prepare products for detecting TIGIT;

[0098] (6) Prepare drugs for treating tumors.

[0099] Preferably, the TIGIT mentioned in (5) is at least one of human TIGIT and monkey TIGIT; more preferably, human TIGIT.

[0100] Preferably, the product described in (5) is at least one of reagents, test plates and kits.

[0101] Preferably, the tumor mentioned in (6) includes: hematologic malignancies, solid tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric tumors, bladder cancer, endometrial cancer, head and neck cancer, and kidney cancer; further, at least one of colorectal cancer and breast cancer.

[0102] In a sixth aspect, the present invention provides a product comprising at least one of the human TIGIT antibody or antigen-binding fragment thereof of the first aspect of the present invention and an immunoconjugate of the fourth aspect of the present invention; said product is at least one of a reagent, a detection plate and a kit.

[0103] Preferably, the product is used to detect TIGIT.

[0104] Preferably, the TIGIT is at least one of human TIGIT and monkey TIGIT; more preferably, it is human TIGIT.

[0105] A seventh aspect of the present invention provides a pharmaceutical composition comprising at least one of (1) to (4);

[0106] (1) The human TIGIT antibody or antigen-binding fragment thereof of the first aspect of the present invention;

[0107] (2) The nucleic acid molecule of the second aspect of the present invention;

[0108] (3) The expression cassette, recombinant vector, or transgenic cell line of the third aspect of the present invention;

[0109] (4) The immunoconjugate of the fourth aspect of the present invention.

[0110] Preferably, the pharmaceutical composition further comprises other antitumor drugs.

[0111] Preferably, the other antitumor drugs comprise at least one of an antimitotic agent, an inhibitor of topoisomerase I or II, a DNA alkylating agent, an antimetabolite, and a targeted drug; more preferably, the other antitumor drugs comprise a targeted drug.

[0112] Preferably, the targeted drug comprises at least one of a small molecule drug and a monoclonal antibody; more preferably, the targeted drug comprises a monoclonal antibody.

[0113] Preferably, the monoclonal antibody comprises at least one of PD-L1 antibody, PD-1 antibody, TIM-3 antibody, CTLA-4 antibody, LAG-3 antibody, and CD-47 antibody; more preferably, the monoclonal antibody comprises PD-L1 antibody.

[0114] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0115] Preferably, the pharmaceutical composition is used to treat tumors.

[0116] Preferably, the tumor includes: hematologic malignancies, solid tumors, non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric tumors, bladder cancer, endometrial cancer, head and neck cancer, and kidney cancer; more preferably, at least one of colorectal cancer and breast cancer.

[0117] Preferably, the conjugation portion of the immunoconjugate is a drug, toxin, and / or therapeutic isotope.

[0118] An eighth aspect of the present invention provides a method for treating tumors, comprising administering to a patient an effective amount of a human TIGIT antibody or antigen-binding fragment thereof of the first aspect of the present invention, a nucleic acid molecule of the second aspect of the present invention, an expression cassette of the third aspect of the present invention, a recombinant vector or transgenic cell line, an immunoconjugate of the fourth aspect of the present invention, and / or a pharmaceutical composition of the seventh aspect of the present invention.

[0119] The beneficial effects of this invention are:

[0120] This invention provides a human TIGIT antibody or its antigen-binding fragment, which has a strong binding ability to hTIGIT and can induce B-hTIGIT transgenic mouse T cells, Jurkat cells, and human PBMC cells to secrete cytokines (IL-2 and / or IFN-γ). It has significant ADCC and CDC effects and can significantly reduce tumor volume / weight in colon cancer CT26 cell line tumor-bearing mice and breast cancer 4T1 cell line tumor-bearing mice without affecting mouse weight (i.e., without side effects), prolonging the survival time of mice. It also has good pharmacokinetic characteristics and can be used to detect hTIGIT and treat tumors.

[0121] Furthermore, by combining the human TIGIT antibody or its antigen-binding fragment provided by the present invention with other antitumor drugs, its antitumor effect is further improved. Attached Figure Description

[0122] Figure 1 This is a graph showing the results of the binding activity of hTIGIT mouse antibodies m60H5 and m51C1 with the antigen hTIGIT-his protein in Example 3.

[0123] Figure 2 This is a graph showing the results of the binding activity of hTIGIT mouse antibodies m60H5 and m51C1 with hTIGIT on the 293F-hTIGIT-short#1B1 cell line in Example 3.

[0124] Figure 3 This is a graph showing the competitive activity of hTIGIT mouse antibodies m60H5 and m51C1 against PVR in Example 4, which binds to hTIGIT on the surface of 293F-hTIGIT-short cells.

[0125] Figure 4 The graphs shown in Example 5 illustrate the binding activity of hTIGIT mouse antibodies m60H5 and m51C1 with monkey and mouse TIGIT proteins: A represents the binding activity of hTIGIT mouse antibodies m60H5 and m51C1 with monkey TIGIT proteins; B represents the binding activity of hTIGIT mouse antibodies m60H5 and m51C1 with mouse TIGIT proteins.

[0126] Figure 5 The following are the results of the binding activity of the humanized hTIGIT antibodies h60H5 and h51C1 with hTIGIT in Example 6: A is the result of the ELISA method detecting the binding activity of the humanized hTIGIT antibodies h60H5 and h51C1 with hTIGIT; B is the result of the flow cytometry (FCM) method detecting the binding activity of the humanized hTIGIT antibodies h60H5 and h51C1 with the hTIGIT protein expressed on the cell surface.

[0127] Figure 6 This is a graph showing the competitive activity of the humanized hTIGIT antibodies h60H5 and h51C1 against PVR in Example 7, which binds to hTIGIT on the surface of 293F-hTIGIT-short cells.

[0128] Figure 7 This is a graph showing the effect of hTIGIT mouse antibodies m60H5 and m51C1 on the level of mIL-2 secretion by T cells in B-hTIGIT transgenic mice in Example 8.

[0129] Figure 8 This is a graph showing the effect of hTIGIT mouse antibodies m60H5 and m51C1 on the level of mIFN-γ secreted by T cells in B-hTIGIT transgenic mice in Example 8.

[0130] Figure 9 This is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the level of mIL-2 secretion by T cells in B-hTIGIT transgenic mice in Example 9.

[0131] Figure 10 This is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the level of mIFN-γ secreted by T cells in B-hTIGIT transgenic mice in Example 9.

[0132] Figure 11 This is a graph showing the effect of hTIGIT mouse antibodies m60H5 and m51C1 on the level of human IL-2 secreted by Jurkat cells in Example 10.

[0133] Figure 12 This is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the level of human IL-2 secreted by Jurkat cells in Example 11.

[0134] Figure 13 This is a graph showing the effect of hTIGIT mouse antibodies m60H5 and m51C1 on the activation and secretion of human IL-2 in human PBMC cells in Example 12.

[0135] Figure 14 This is a graph showing the effect of hTIGIT mouse antibodies m60H5 and m51C1 on the activation and secretion of human IFN-γ in human PBMC cells in Example 12.

[0136] Figure 15 This is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the level of human IFN-γ secretion activated by human PBMC cells in Example 13.

[0137] Figure 16 This is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the activation and secretion of human IL-2 in human PBMC cells in Example 13.

[0138] Figure 17 This is a graph showing the competition between hTIGIT murine antibodies m60H5, m51C1 and 4.1D3 for hTIGIT epitope binding in Example 14.

[0139] Figure 18The ADCC effect diagrams of the humanized hTIGIT antibodies h60H5 and h51C1 in Example 15 are shown below: A is the ADCC effect diagram of the humanized hTIGIT antibody h60H5; B is the ADCC effect diagram of the humanized hTIGIT antibody h51C1.

[0140] Figure 19 The following are CDC effect diagrams of the humanized hTIGIT antibodies h60H5 and h51C1 in Example 16: where A is the CDC effect diagram of the humanized hTIGIT antibody h60H5; and B is the CDC effect diagram of the humanized hTIGIT antibody h51C1.

[0141] Figure 20 The following is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on tumor-bearing mice with CT26 colon cancer cells in Example 17: A shows the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the tumor volume of tumor-bearing mice with CT26 colon cancer cells; B shows the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the body weight of tumor-bearing mice with CT26 colon cancer cells.

[0142] Figure 21 This is a survival curve of mice bearing colon cancer CT26 cell line treated with the humanized hTIGIT antibodies h60H5 and h51C1 in Example 17.

[0143] Figure 22 The results of the tumor re-inoculation challenge experiment in tumor-bearing cured mice in Example 18 are shown in Figure A, which is the drug efficacy (tumor size)-drug concentration curve after re-challenge of CT26 in humanized mouse BALB / C-huTIGIT colon cancer cells; and Figure B is the body weight change curve after re-inoculation of CT26 in humanized mice.

[0144] Figure 23 The following is a graph showing the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on 4T1 breast cancer cells-bearing B-huTIGIT mice in Example 19: A shows the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the tumor volume of 4T1 breast cancer cells-bearing B-huTIGIT mice; B shows the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the tumor weight of 4T1 breast cancer cells-bearing B-huTIGIT mice; and C shows the effect of the humanized hTIGIT antibodies h60H5 and h51C1 on the body weight of 4T1 breast cancer cells-bearing B-huTIGIT mice.

[0145] Figure 24This is a survival curve of the humanized hTIGIT antibodies h60H5 and h51C1 used in Example 19 to treat 4T1 breast cancer cells bearing B-huTIGIT mice.

[0146] Figure 25 This is a pharmacokinetic diagram of the hTIGIT humanized antibodies h60H5 and h51C1 in Example 20.

[0147] Figure 26 The following are graphs showing the effects of the humanized hTIGIT antibodies h60H5 and h51C1 combined with PD-L1 antibody on the volume of CT26 cell xenografts in Example 21: A is the effect of the humanized hTIGIT antibody h51C1 combined with atezolizumab on the volume of CT26 cell xenografts; B is the effect of the humanized hTIGIT antibody h60H5 combined with atezolizumab on the volume of CT26 cell xenografts.

[0148] Figure 27 This is a schematic diagram illustrating the signal transduction mechanism of TIGIT protein in T cells and NK cells. Detailed Implementation

[0149] The present invention will be further described in detail below through specific embodiments.

[0150] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0151] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0152] Example 1: Preparation of human TIGIT mouse antibody

[0153] The human TIGIT mouse monoclonal antibody was produced using hybridoma technology, as detailed below:

[0154] Balb / c mice were immunized using hTIGIT (NP_776160.2) as the antigen. The immunized mice underwent three immunizations with purified antigen and complete Freund's adjuvant, and the immune response was assessed after blood collection via the tail vein. Serum samples were screened using ELISA and flow cytometry to obtain mice containing human TIGIT immunoglobulin. Spleen cells from mice with the highest levels of anti-TIGIT immunoglobulin were fused with mouse myeloma cells SP2 / 0 (ATCC ID CRL-1581). The resulting hybridoma cells were screened for antibodies, yielding mouse human TIGIT antibodies: m60H5 and m51C1. The mouse human TIGIT antibodies m60H5 and m51C1 were then sequenced.

[0155] The amino acid sequence of the heavy chain variable region of m60H5 is EVQLQQSGPELVKPGASLKISCKTS GYTFTEYT MHWVKQSHGKSLEWIGG INPNNGGT KFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYC ARSGNWDYAMDY WGQGTSVTVSS (SEQ ID NO.1, where the underlined portions are CDR1, SEQ ID NO.2, CDR2, SEQ ID NO.3, CDR3, and SEQ ID NO.4 respectively), encodes the amino acid sequence as shown in SEQ ID NO. The nucleotide sequence of the heavy chain variable region of m60H5 shown in NO.1 is: GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCACTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAAGATCGGGGAACTGGGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO.5); the amino acid sequence of the light chain variable region of m60H5 is: DIVMTQSHKFMSTSVGDRVSITCKAS QHVSTA VVWYQQKPGQSPKLLIY SASYRYTGVDRFTGSGSGTDFTFTISSVQAEDLAVYYC QQHYITPWT FGGGTKLEIKRADA (SEQ ID NO.6, where the underlined portions are CDR1, SEQ ID NO.7, CDR2, SEQ ID NO.8, CDR3, and SEQ ID NO.9 in sequence), encodes the amino acid sequence shown in SEQ ID NO.6. The nucleotide sequence of the light chain variable region of m60H5 shown in IDNO.6 is: GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAACATGTGAGTACTGCTGTAGTCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCGT ACCGGTACACTGGAGTCCCTGATCGGTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAGGACCTGGCAGTTTTATTACTGTCAGCAACATTATATTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGAT (SEQ ID NO. 10).

[0156] The amino acid sequence of the heavy chain variable region of m51C1 is QVQLQQSGAELVKPGASVKLSCKAS GYTFTEYF IHWIKQKSGQGLEWIGW FYPGSGSI KYNERFKDKATLTADKSSSTVYMELSRLTSEDSAVYFC ARHEMRYGNYVLDYWGQGTTLTVSS (SEQ ID NO.11, where the underlined portions are CDR1, SEQ ID NO.12, CDR2, SEQ ID NO.13, CDR3, and SEQ ID NO.14 respectively), encodes the amino acid sequence as shown in SEQ ID NO. The nucleotide sequence of the heavy chain variable region of m51C1 shown in NO.11 is: CAGGTCCAGCTGCAGCAGTCAGGAGCTGAGCTGGTGAAACCCGGGGCATCAGTGAAGCTGTCCTGTAAGGCTTCTGGCTACACCTTCACTGAGTATTTTATACACTGGATAAAGCAGAAGTCTGGACAGGGTCTTGAGTGGATTGGGTGGTTTTACCCTGGAAGTGGTAGTATAAAGTACAATGAGAGATTCAAGGACAAGGCCACATTGACTGCGGACAAATCCTCCAGCACAGTCTATATGGAGCTTAGTAGATTGACATCTGAAGACTCTGCGGTCTATTTCTGTGCAAGACACGAGATGAGGTATGGTAACTACGTCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO.15); the amino acid sequence of the light chain variable region of m51C1 is: QAVVTQESALTTSPGETVTLTCRSS TGAVT TRNY ANWVQEKPDHLFTGLIG GTN NRVPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC GLWYSNHLVFGGGTKLTVLGQPK (SEQ ID NO.16, where the underlined portions are CDR1, SEQ ID NO.17, CDR2, SEQ ID NO.18, CDR3, and SEQ ID NO.19 in sequence), encodes the amino acid sequence as shown in SEQ ID NO. The nucleotide sequence of the light chain variable region of m51C1 shown in NO.16 is: CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGAAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACAGGTCTAATAGGTGGTAC CAACAACCGAGTTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATTTATTTCTGTGGTCTATGGTACAGCAACCATTTGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAG(SEQ ID NO.20).

[0157] Example 2: Preparation of humanized TIGIT antibody

[0158] Referring to the light chain variable region and heavy chain variable region sequences of the m60H5 and m51C1 antibodies, the humanized templates that best match their non-CDR regions were selected. The CDR regions of the murine antibodies were transplanted onto the selected humanized templates, replacing the CDR regions of the human templates, to obtain the humanized antibodies h60H5 and h51C1. Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on the embedded residues, residues that directly interact with the CDR region, and residues that have an important influence on the conformation of VL and VH to obtain the humanized antibody. The sequence of the heavy chain variable region of the humanized antibody h60H5 is: QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGQRLEWIGGINPNNGGTSYNQKFQGRVTITVDTSASTAYMELSSLRSEDTAVYYCARSGNWDYAMDYWGQGTTVTVSS (SEQ ID NO: 21), and the sequence of the light chain variable region of the humanized antibody h60H5 is: DIQMTQSPSSMSASVGDRVTITCKASQHVSTAVWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYITPWTFGGGTKLEIKRTVA (SEQ ID NO: 21). NO: 22); The sequence of the heavy chain variable region of the humanized antibody h51C1 is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYFIHWVRQAPGQGLEWIGWFYPGSGSIKYNERFKDRVTLTADTSISTAYMELSRLRSDDTAVYYCARHEMRYGNYVLDYWGQGTTVTVSS (SEQ ID NO: 23); The sequence of the light chain variable region of the humanized antibody h51C1 is: QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTRNYANWVQQKPGQAPRGLIGGTNNRVPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCGLWYSNHLVFGGGTKLTVLGQPKA (SEQ ID NO: 24).

[0159] Example 3: Binding activity of hTIGIT murine antibody to hTIGIT

[0160] The binding activity of hTIGIT mouse antibodies m60H5 and m51C1 and positive control PcAb (4.1D3, purchased from Sanyou Biotechnology Co., Ltd., WJ20201031) to hTIGIT-His protein was detected by ELISA. hTIGIT-His protein was diluted to 0.5 μg / mL with CBS buffer, and 50 μL / well was coated onto each well of an ELISA plate and incubated overnight at 4°C. The plates were washed three times with PBST buffer (200 μL / well) and blotted dry. Then, 200 μL of 1% BSA (dissolved in PBS) was added to each well, and the plates were incubated at 37°C for 2 hours. After incubation, wash the plate three times with PBST, 200 μL / well, and blot dry. Dilute the positive control PcAb (4.1D3) and hTIGIT mouse antibodies m60H5 and m51C1 to 1 μg / mL with PBST. Perform serial dilutions in the dilution plate from column 1 to 11 using a 3-fold dilution method, for a total of 11 groups. Group 12 contains 1×PBST as a blank control. Each antibody concentration is tested in duplicate. Transfer 100 μL to the ELISA plate using a multipipe and incubate at 37°C for 1 h. After incubation, wash the plate three times with PBST, 200 μL / well, and blot dry. Dilute the HRP-labeled anti-mouse and anti-human IgG antibodies (both diluted 1:5000) with 1% BSA (dissolved in PBST), 100 μL / well, and incubate at 37°C for 1 h. After incubation, wash the plate three times with PBST (200 μL / well) and blot dry. Add 50 μL of TMB chromogenic solution to each well for colorimetric reaction, and incubate in the dark for 5 min. After 5 min of incubation, add 50 μL of hydrochloric acid stop solution directly to stop the reaction. Measure the absorbance at 450 nm (OD450) using an ELISA reader in absorbance mode. ELISA results are shown below. Figure 1 As shown, the murine hTIGIT antibodies m60H5 and m51C1, along with the positive control PcAb (4.1D3), exhibit strong binding activity to the hTIGIT-His protein. The obtained murine antibodies m60H5 (EC50 = 0.00277 μg / mL) and m51C1 (EC50 = 0.00396 μg / mL) show similar binding activity to the positive control 4.1D3 (EC50 = 0.00255 μg / mL).

[0161] The 293F-hTIGIT-short#1B1 cell line was constructed as follows: The extracellular segment and transmembrane region gene sequences of TIGIT were synthesized (Gene ID: 201633, synthesized by Anhui General Biotechnology Co., Ltd.). These sequences were then inserted between the EcoRI and BamHI regions of the pLVX-Puro plasmid. The recombinant plasmid was transfected into 293F cells (purchased from ATCC), and a stable cell line expressing TIGIT protein was formed through selection using cell culture medium containing 2.5 μg / mL puromycin. The cell line was then cloned using a limiting dilution method. After screening and identification, clone #1B1 was determined to be a stable monoclonal cell line expressing the extracellular segment of TIGIT.

[0162] The 293F-hTIGIT-short#1B1 cell line was digested, terminated, centrifuged, resuspended, and counted to adjust the cell density to 4 × 10⁻⁶ cells / year. 6 Count / mL, at 50μL / tube, take 20×10 4 Cells were added to 1.5 mL EP tubes. Prepared hTIGIT mouse antibodies m60H5 and m51C1, and positive control PcAb (4.1D3) were added. hTIGIT mouse antibodies and PcAb (4.1D3) were diluted to an initial concentration of 12 μg / mL, and serially diluted 1:3 to seven concentrations. 150 μL of each solution was added to the corresponding tube. A blank control was prepared by adding 150 μL of PBS. Each antibody concentration was administered in a single tube. The cells were gently mixed by pipetting and incubated on ice for 1 h. After incubation, each cell was washed once with 500 μL of PBS and centrifuged at 2000 rpm for 5 min. The supernatant was discarded. Add fluorescent secondary antibody (Alexa 488 fluorescently labeled anti-mouse and anti-human IgG antibodies diluted 1:500 with PBS), 200 μL / tube to the corresponding tube, gently pipette to mix, and incubate on ice in the dark for 30 min. After 30 min incubation, signal detection was performed using flow cytometry. The binding activity of hTIGIT mouse antibody and positive control PcAb (4.1D3) was analyzed, and EC50 was obtained for each. The surface hTIGIT binding activity of hTIGIT mouse antibody and positive control PcAb (4.1D3) on 293F-hTIGIT-short cells was detected by flow cytometry (FCM). The FCM results are shown below. Figure 2As shown, the murine hTIGIT antibodies m60H5 (EC50 = 0.631 μg / mL) and m51C1 (EC50 = 0.811 μg / mL) and the positive control PcAb (4.1D3, EC50 = 1.11 μg / mL) have a strong binding ability to hTIGIT on the cell surface. The binding activities of murine antibodies m60H5 and m51C1 and the positive control are comparable.

[0163] Example 4: Competitive binding activity of hTIGIT mouse antibody

[0164] The 293F-hTIGIT-short#1B1 cell line was digested, terminated, centrifuged, resuspended, and counted to adjust the cell density to 4 × 10⁻⁶ cells / year. 6 Count / mL, at 50μL / tube, take 20×10 4 Cells were added to 1.5 mL EP tubes. hTIGIT mouse antibodies m60H5 and m51C1, and the positive control PcAb (4.1D3) were diluted to an initial concentration of 108 μg / mL, and seven concentrations were obtained using a 1:3 serial dilution method. Each antibody concentration was added to a single tube at a rate of 50 μL / tube. 50 μL of PBS was added to the blank group, the PVR-hFc group (containing hTIGIT mouse antibodies m60H5 and m51C1), and the PVR-mFc group (containing PcAb (4.1D3)). The mixture was gently pipetted and incubated on ice for 15 min. After 15 min, the PVR-hFc group was treated with 6 μg / mL PVR-hFc protein (sequence as shown in SEQ ID NO. 25), and the PVR-mFc group was treated with 6 μg / mL PVR-mFc protein (sequence as shown in SEQ ID NO. 26), 100 μL / tube. The blank group was treated with 100 μL PBS, gently pipetted to mix, and incubated on ice for another 1 h. After incubation, each tube was washed once with 500 μL PBS by centrifugation at 2000 rpm for 5 min, and the supernatant was discarded. Alexa 488 fluorescently labeled anti-mouse and anti-human IgG antibodies were diluted with PBS (1:500), 200 μL / tube, gently pipetted to mix, and incubated on ice in the dark for 30 min. After 30 min of incubation, the signal was detected by flow cytometry. Based on the antagonistic activity results of murine antibodies m60H5 and m51C1, both antibodies exhibited similar antagonistic activity to 4.1D3. Figure 3 The competitive activity of each antibody was concentration-dependent. The IC50 values ​​for m60H5, m51C1, and 4.1D3 were 1.54 μg / mL, 1.35 μg / mL, and 1.27 μg / mL, respectively.

[0165] Example 5: Cross-binding activity of hTIGIT mouse antibody with monkey and mouse TIGIT proteins

[0166] The binding activities of hTIGIT mouse antibodies m60H5 and m51C1 and the positive control PcAb (4.1D3) to monkey (accession number: XP_016797180.2) or mouse TIGIT protein (accession number: NP_001139797.1) were analyzed using an indirect ELISA method. The method was the same as the ELISA detection method in Example 3, except that mouse TIGIT-his protein and monkey TIGIT-his protein were diluted to 0.5 μg / mL with CBS buffer, and each antibody concentration was tested in duplicate. Homology analysis of human, monkey, and mouse TIGIT protein sequences showed high homology with human and monkey TIGIT proteins, but low homology with mouse TIGIT proteins. Results are as follows: Figure 4 As shown: the murine hTIGIT antibodies m60H5, m51C1, and the positive control PcAb (4.1D3) largely maintained their binding activity to monkey TIGIT protein. In particular, m60H5 and the positive control PcAb (4.1D3) showed high binding activity to monkey TIGIT protein, while m51C1 had a weak affinity for monkey TIGIT protein. The murine hTIGIT antibodies m60H5, m51C1, and the positive control PcAb (4.1D3) all had very weak binding affinity to murine TIGIT protein and can be considered as having no affinity.

[0167] Example 6: Binding activity of hTIGIT humanized antibody to hTIGIT

[0168] The binding activities of the humanized hTIGIT antibodies h60H5 and h51C1 and the positive control PcAb (4.1D3) to the hTIGIT protein were detected by ELISA and flow cytometry, respectively. The methods were the same as in Example 3, except that the mouse hTIGIT antibodies m60H5 and m51C1 were replaced with the humanized hTIGIT antibodies h60H5 and h51C1. For the ELISA detection method, 100 μL of HRP-labeled anti-human IgG antibody (1:5000 dilution) was diluted with 1% BSA (dissolved in PBST), with two replicates per antibody concentration. For the flow cytometry detection method, fluorescent secondary antibody (Alexa 488 fluorescently labeled anti-human IgG antibody diluted 1:500 with PBS) was added to the corresponding tubes at 200 μL per tube, gently pipetted to mix, and incubated on ice in the dark for 30 min, with one tube per antibody concentration. The ELISA results are as follows: Figure 5As shown in Figure A: the humanized hTIGIT antibodies h60H5 (EC50 = 0.006 μg / mL), h51C1 (EC50 = 0.007 μg / mL), and the positive control PcAb (4.1D3, EC50 = 0.003 μg / mL) all exhibited strong binding activity to the hTIGIT-His protein; FCM detection results are as follows. Figure 5 As shown in Figure B, the humanized hTIGIT antibodies h60H5 (EC50 = 1.57 μg / mL), h51C1 (EC50 = 2.98 μg / mL), and the positive control PcAb (4.1D3, EC50 = 1.05 μg / mL) have a strong binding ability to hTIGIT on the cell surface, and the binding activities of the three are comparable.

[0169] Example 7 Competitive binding activity of hTIGIT humanized antibody

[0170] The ability of humanized hTIGIT antibodies h60H5 and h51C1 and the positive control PcAb (4.1D3) to compete with hTIGIT for binding to the poliovirus receptor (PVR) and TIGIT was detected by flow cytometry (method as in Example 4), except that the murine hTIGIT antibodies m60H5 and m51C1 were replaced with humanized hTIGIT antibodies h60H5 and h51C1. Alexa 488 fluorescently labeled anti-human IgG antibodies were diluted 1:500 with PBS, and 200 μL / tube was added to the corresponding tubes. The tubes were gently pipetted to mix, and incubated on ice in the dark for 30 min. Each antibody concentration was counted in a single tube. Results are as follows: Figure 6 As shown, the humanized hTIGIT antibodies h60H5 (IC50 = 2.00 μg / mL), h51C1 (IC50 = 5.21 μg / mL), and the positive control PcAb (4.1D3, IC50 = 3.77 μg / mL) all exhibited strong competitive activity against PVR. In particular, the competitive activity of h60H5 was superior to that of h51C1 and the positive control PcAb (4.1D3).

[0171] Example 8: hTIGIT mouse antibody induces T cells of B-hTIGIT transgenic mice to secrete cytokines.

[0172] B-hTIGIT transgenic mice (purchased from Nanjing Jicui Pharmaceutical Co., Ltd.) were euthanized by enucleation and cervical dislocation. The spleen was then aseptically removed, and the surface mucosa and other tissues were removed. The spleen was placed on a 70μm sieve, moistened with serum-free DMEM, minced, and then ground using the core of a 2.5mL sterile syringe. The sieve was then rinsed with 3mL of serum-free DMEM until the spleen tissue was thoroughly ground. The cell suspension was then transferred to a 40μm sieve and filtered again using a 10mL pipette. The filtered cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were washed once with 3mL of PBS, centrifuged again at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in 1mL of DMEM complete medium (containing 10% FBS) and transferred to a T75 flask. DMEM complete medium was added to a final volume of 15mL, and the cells were cultured in a 37℃, 5% CO2 incubator. Anti-mouse CD3e protein was coated at a concentration of 10 μg / mL (anti-mouse CD3e protein was diluted to 10 μg / mL with sterile PBS, and 50 μL / well was added to a 96-well plate, sealed with adhesive tape, and incubated overnight at 4°C). The supernatant of the overnight-coated Anti-mouse CD3e protein in the 96-well plate was discarded, the plate was blotted dry, and then washed once with sterile PBS, discarding the supernatant and blotting dry again. Mouse spleen cells were centrifuged, resuspended, and counted, and the cell density was adjusted to 2 × 10⁶ cells / well. 6 Cells / mL, at a rate of 0.1 × 10⁻⁶ 6Cells were added at 50 μL / well to 96-well plates. Corresponding antibody concentrations were prepared: hTIGIT mouse antibodies m60H5, m51C1, and PcAb (4.1D3). All antibodies were initially diluted at a concentration of 40.5 μg / mL (1.5×), and then serially diluted 1:3 to three concentrations using DMEM complete medium (containing 10% FBS). Antibody and induction groups (with an equal volume of PBS) were added to wells at 100 μL / well, while the blank group was added to wells at 100 μL / well. Each concentration was tested in duplicate. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 48 h. After 48 h of incubation, the cells in the 96-well plates were centrifuged at 2000 rpm for 5 min. Cell supernatant was collected, and mouse cytokines IL-2 and IFN-γ were detected according to the instructions of the Mouse IL-2 precoated ELISA kit and the Mouse IFN-γ precoated ELISA kit. The absorbance at 450 nm (OD450) was measured using an ELISA reader in absorbance mode. GraphPad Prism 5 data processing software was used to obtain bar graphs showing the levels of mouse IFN-γ and mouse IL-2 expressed in spleen cells, using the positive control PcAb (4.1D3), hTIGIT mouse antibodies m60H5, and m51C1. The experimental results are shown below. Figure 7 , 8 As shown: the positive control PcAb (4.1D3), hTIGIT murine antibody m60H5, and m51C1 showed different abilities to induce T cells to secrete mIFN-γ and mIL-2; all murine antibodies could continuously induce T cells to secrete mIFN-γ ( Figure 8 In particular, m51C1 and m60H5 exhibit very strong sustained inducing activity; while their effect on mIL-2 secretion is not significant. Figure 7 ).

[0173] Example 9: hTIGIT humanized antibody induces T cells of B-hTIGIT transgenic mice to secrete cytokines.

[0174] The processing method in this embodiment is the same as in Example 8, except that the hTIGIT mouse antibodies m60H5 and m51C1 are replaced with hTIGIT humanized antibodies h60H5 and h51C1, with two replicates per group per concentration. The results are as follows: Figure 9 , 10 As shown: Positive control PcAb (4.1D3), humanized hTIGIT antibody h60H5, and h51C1 can all induce T cells of B-hTIGIT transgenic mice to secrete IFN-γ and IL-2, and the inducing activity of humanized hTIGIT antibody h60H5 and h51C1 at some concentrations is better than that of positive control PcAb (4.1D3).

[0175] Example 10: hTIGIT mouse antibody induces Jurkat cells to activate and secrete cytokines

[0176] Jurkat cells were collected and counted, and the cell density was adjusted to 0.8 × 10⁻⁶. 6 Cells / mL, at a rate of 0.04 × 10⁻⁶ 6 Plate 96-well plates with 50 μL / well. Add the corresponding prepared antibodies: hTIGIT mouse antibodies m60H5, m51C1, and positive control PcAb (4.1D3), all diluted starting at an initial concentration of 13.5 μg / mL (i.e., 1.5 times the initial concentration), and serially diluted 1:3 to three concentrations: 9, 3, and 1 μg / mL. The dilution buffer is 1640 complete medium (containing 10% FBS) containing 0.375 μg / mL (1.5×) CD3 protein and 0.1875 μg / mL (1.5×) CD28 protein. The induction group is treated with 0.375 μg / mL CD3 protein and 0.1875 μg / mL... CD28 protein was added to 1640 complete medium, and a blank control group was added to 1640 complete medium, 100 μL / well was added to each cell well, with two replicates per concentration for each group. The cells were incubated at 37℃ in a 5% CO2 cell culture incubator for 48 h. After 48 h, the cells in the 96-well plate were centrifuged at 2000 rpm for 5 min, and the supernatant was collected. Human cytokine IL-2 was detected according to the Human IL-2 precoated ELISA kit instructions. The absorbance at 450 nm (OD450) was measured using an ELISA reader. The levels of human IL-2 expressed in Jurkat cells stimulated with the positive control PcAb (4.1D3) and hTIGIT mouse antibody were plotted and analyzed using GraphPad Prism 5 data processing software. Results are shown below. Figure 11 As shown, hTIGIT murine antibodies m60H5 and m51C1 can induce activated Jurkat cells to continuously secrete human IL-2 cytokine, while the positive control 4.1D3 lacks the ability to continuously induce activation.

[0177] Example 11: hTIGIT humanized antibody induces Jurkat cells to activate and secrete cytokines

[0178] The processing method in this embodiment is the same as in Example 10, except that the hTIGIT mouse antibodies m60H5 and m51C1 are replaced with hTIGIT humanized antibodies h60H5 and h51C1, with two replicates per group per concentration. The results are as follows: Figure 12As shown, the humanized hTIGIT antibodies h60H5, h51C1 and positive control 4.1D3 have limited ability to induce sustained secretion of human IL-2 cytokine in activated Jurkat cells; compared with mouse antibodies, the ability of each humanized antibody to induce IL-2 secretion is weaker, which may also be related to the human IgG1 Fc functional domain.

[0179] Example 12 hTIGIT mouse antibody induces human PBMC cells to secrete cytokines

[0180] The purified PBMC cells were counted, and the cell density was adjusted to 2 × 10⁶ cells / year. 6 Cells / mL, at a rate of 0.1 × 10⁻⁶ 6 Cells were seeded in 96-well plates at 50 μL / well. The corresponding prepared antibodies, hTIGIT mouse antibody and positive control PcAb (4.1D3), were added, both starting at an initial concentration of 40.5 μg / mL (1.5X) and serially diluted 1:3 to three concentrations. The diluent was DMEM complete medium (containing 10% FBS) containing 0.75 μg / mL (1.5×) CD3 protein and 0.375 μg / mL (1.5×) CD28 protein. The induction group received only DMEM complete medium containing 0.75 μg / mL CD3 protein and 0.375 μg / mL CD28 protein, while the control group received DMEM complete medium. 100 μL / well was used for each group, with two replicates per concentration. Cells were incubated at 37°C in a 5% CO2 cell culture incubator for 24 h. Twenty-four hours later, the cell culture plates were centrifuged at 2000 rpm for 5 minutes, and the cell supernatant was collected. Human IL-2 and human IFN-γ were detected according to the instructions of the Human IL-2 precoated ELISA kit and the Human IFN-γ precoated ELISA kit. The absorbance at 450 nm (OD450) was measured using an ELISA reader. GraphPad Prism 5 data processing software was used to plot and analyze the levels of human IFN-γ and human IL-2 expressed in PBMCs induced by the positive control PcAb (4.1D3) and hTIGIT mouse antibody. The ability of hTIGIT mouse antibody and the positive control 4.1D3 to continuously secrete human IFN-γ and IL-2 in human PBMCs co-stimulated with CD3 and CD28 was detected. The results are as follows. Figure 13 , 14 As shown: both hTIGIT murine antibody and positive control 4.1D3 can induce sustained IL-2 secretion. Figure 13 However, its induction of human IFN-γ secretion levels was not very significant. Figure 14 ).

[0181] Example 13: hTIGIT humanized antibody induces human PBMC cells to secrete cytokines

[0182] The processing method in this embodiment is the same as in Example 12, except that the hTIGIT mouse antibodies m60H5 and m51C1 are replaced with hTIGIT humanized antibodies h60H5 and h51C1, with two replicates per group per concentration. The results are as follows: Figure 15 , 16 As shown: hTIGIT humanized antibodies h60H5 and h51C1 can induce sustained secretion of IFN-γ. Figure 15 However, its ability to continuously secrete IL-2 in humans is relatively weak.

[0183] Example 14 Analysis of hTIGIT Epitope Competition between Mouse hTIGIT Antibody and 4.1D3

[0184] Cell collection and counting: The 293F-hTIGIT-short#1B1 cell line was digested, terminated, centrifuged, resuspended, and counted. The cell density was adjusted to 4 × 10⁻⁶ cells / year. 6 Cells / mL, 50μL / tube, for a total of 20×10 4Cells were added to 1.5 mL EP tubes. Prepared hTIGIT mouse antibodies m60H5 and m51C1 were added. Both hTIGIT mouse antibodies m60H5 and m51C1 were started at a concentration of 108 μg / mL and serially diluted 1:3 to seven concentrations, with 50 μL added to each tube. For the control group, PcAb(4.1D3) group, and hTIGIT mouse antibody group, 50 μL of PBS was added, and the mixture was gently pipetted to mix. Each group and concentration was placed in a single tube and incubated on ice for 15 min. After 15 min, 100 μL of prepared PcAb(4.1D3) (6 μg / mL) antibody was added to the hTIGIT mouse antibody group and the PcAb(4.1D3) group, and 100 μL of PBS was added to the control group. The mixture was gently pipetted to mix, and the tubes were incubated on ice for another 1 h. After incubation, add 500 μL of PBS and wash once by centrifugation. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Add 200 μL of fluorescent secondary antibody (Alexa 488 fluorescently labeled anti-human IgG antibody diluted 1:500 with PBS) to each tube, gently pipette to mix, and incubate on ice in the dark for 30 min. After 30 min of incubation, analyze the samples using flow cytometry. Analyze the binding of hTIGIT murine antibody to the hTIGIT epitope of PcAb (4.1D3) to obtain the competitive binding of m60H5 and m51C1, and calculate their IC50 values. Perform competitive binding activity analysis between the hTIGIT murine antibody and the 4.1D3 antibody (human IgG1 subtype) against the hTIGIT epitope to determine the selected hTIGIT murine antibodies m60H5 (IC50 = 5.02 μg / mL) and m51C1 (IC50 = 4.08 μg / mL) and the positive control PcAb (4.1D3). Results are as follows. Figure 17 As shown: From the epitope binding curve and the fluorescence intensity-concentration-dose relationship, it can be seen that the screened hTIGIT mouse antibodies m60H5 and m51C1 compete with the 4.1D3 antibody for the same or very close antigenic epitopes of hTIGIT.

[0185] Example 15 ADCC effect of hTIGIT humanized antibody

[0186] Prepare 293F-TIGTI-short#1B1 cells and Jurkat-NFAT-Luc2-CD16a-V158 cells (purchased from Beijing Kangyuan Bocheng Co., Ltd., #KC-1507). Count the cells of both lines and adjust the cell density to 0.4 × 10⁻⁶. 6 Cells / mL. The two cell lines were mixed at a 1:1 ratio, and the mixed cells were seeded at 50 μL / well in a 96-well white (opaque) plate, i.e., 0.02 × 10⁶ cells from each cell line. 6Cells were added to each well. The corresponding prepared antibodies, hTIGIT humanized antibodies h60H5 and h51C1, and the positive control PcAb (4.1D3), were all initially diluted at a concentration of 2 μg / mL (2×), and then serially diluted 11 times at a 1:3 ratio. The dilution medium was 1640 complete medium (containing 10% FBS). For the blank control, 50 μL of 1640 complete medium was added to each well of a 96-well plate. Each antibody concentration was used in duplicate. Cells were incubated at 37°C in a 5% CO2 cell culture incubator for 6 hours. After 6 hours of co-incubation, the 96-well plates were removed and allowed to equilibrate at room temperature for 15 minutes. Then, 50 μL of luciferase substrate reagent was added to each well, and the reaction was carried out at room temperature in the dark for 5 minutes. The chemiluminescence signal was detected using a microplate reader in luminescence mode. Data processing software was used to obtain four-parameter curves of the fluorescence signals of the positive control PcAb (4.1D3) and the humanized hTIGIT antibody, thereby obtaining the EC50 value of antigen-antibody-dependent cell killing. The chemiluminescent reporter gene approach was used to simulate antibody-dependent cell killing effects of T cells or NK cells. This was achieved by constructing Jurkat cells that exogenously expressed the FcγRIII (CD16) receptor and its downstream NFAT transcription factor, along with the binding domain of this transcription factor and a luciferase reporter gene capable of transcription, to represent the ADCC effect. When the constructed Jurkat-NFAT-Luc2-CD16a cells were co-incubated with 293F-TIGIT-short cells expressing hTIGIT, the antibody activated the signal transduction pathway in the Jurkat-NFAT-Luc2-CD16a cells under antibody-mediated action. Experimental results are as follows: Figure 18 As shown: hTIGIT humanized antibody and positive control PcAb (4.1D3) both showed significant ADCC effects, while the non-specific antibody group did not show ADCC effects; in addition, h60H5 had a better ADCC effect than h51C1; h60H5 had an effect comparable to positive control 4.1D3.

[0187] Example 16: CDC effect of hTIGIT humanized antibody

[0188] 293F-hTIGIT-short#1B1 cells were collected, counted, and the cell density was adjusted to 0.5 × 10⁻⁶. 6 Cells were seeded at a rate of 40 μL / well in a 96-well plate, resulting in a cell count of 0.02 × 10⁶ cells / mL. 6Cells / well. Add the corresponding prepared antibodies: hTIGIT humanized antibodies h60H5 and h51C1, and the positive control PcAb (4.1D3). All antibodies were initially diluted at a concentration of 400 μg / mL (2×), and then serially diluted 1:2 to two concentrations using Freestyle 293 complete medium (containing 5% FBS). The negative control and strong positive control were in Freestyle 293 complete medium (containing 5% FBS) (the strong positive control was supplemented with the cell lysis reagent prepared in the Cytotoxicity LDH Assay Kit as a means of cell death). For the antibody group, negative control, and strong positive control, add 50 μL / well, with three replicates per concentration for each group. Then incubate the cells at 37°C in a 5% CO2 cell culture incubator for 0.5 h. After 0.5 h, add 10 μL / well of fresh human serum, and then incubate the cells at 37°C in a 5% CO2 cell culture incubator for another 3 h. After 2.5 h of incubation, the strong positive control group was incubated for another 0.5 h at 37 °C in a 5% CO2 cell culture incubator with Lysis Buffer (10 μL / well) from the Cytotoxicity LDH Assay Kit. After 3 h, the pre-prepared Working Solution (100 μL / well) from the Cytotoxicity LDH Assay Kit was added, and the reaction was carried out at room temperature in the dark for 30 min. Then, the Stop Solution (50 μL / well) from the Cytotoxicity LDH Assay Kit was added, and the absorbance at 490 nm (OD490) was immediately measured using a microplate reader. The levels of LDH (serum lactate dehydrogenase) mediated by the negative control group, strong positive control group, positive control group, and hTIGIT humanized antibody were plotted and analyzed using GraphPad Prism 5 data processing software. When performing complement-dependent antibody killing (CDC) assays, fresh anticoagulated human plasma is required to ensure complement activity in the plasma. Experimental results are as follows: Figure 19 As shown, the humanized hTIGIT antibodies h60H5 and h51C1 and the positive control PcAb (4.1D3) all had CDC killing effects on hTIGIT-expressing cells, and the humanized hTIGIT antibodies h60H5 and h51C1 were more effective than the positive control PcAb (4.1D3).

[0189] Example 17: Pharmacodynamic experiment of hTIGIT humanized antibody against CT26 colon cancer cell line tumor-bearing mice.

[0190] Antibody efficacy studies were conducted using 8-12 week old adult Balb / c-huTIGIT (B-huTIGIT) transgenic female mice (purchased from Nanjing Jicui Pharmaceutical Co., Ltd.). After purchase, the transgenic mice were observed and housed in an SPF-grade animal facility, acclimatized for one week, under constant temperature (23±2)℃, humidity 40%–60%RH, and artificial light with 12 hours of illumination and 12 hours of darkness. Co60-irradiated feed and tap water were provided on-site. Balb / c mouse colon cancer cells (CT26.WT) in good growth condition were digested and collected, and the density of the single-cell suspension was adjusted to 1×10⁻⁶. 7 1 × 10⁻⁶ cells / mL. Administered subcutaneously (sc) to each mouse in the axillary region. 6 Cells / 100μL / mouse. After tumor cell inoculation, mice were continued to be fed, and their body weight and tumor volume were continuously monitored until the tumor volume reached 80-120 mm. 3 Subsequently, as a successful tumor-bearing mouse model, mice with successfully developed tumors were randomly divided into groups. A negative control group, a positive control group, and a test drug experimental group were set up according to experimental requirements. Mice in each group were weighed, and the drug groups were further divided into high-dose and low-dose groups, followed by intraperitoneal injection. Changes in mouse body weight and tumor volume were continuously monitored during drug administration until the experimental endpoint. General condition, mental state, activity level, bleeding at the injection site, ulceration, etc., were closely observed, and abnormal conditions of the mice were photographed and recorded. When the tumor volume of the mice reached 3000 mmHg... 3 When mice become extremely emaciated and weak, or exhibit poor vital signs, the experimental endpoint is considered reached, and the mice are euthanized by cervical dislocation. Upon reaching the experimental endpoint, the mice are euthanized by cervical dislocation. Tumor dissection is then photographed and weighed, and drug efficacy (tumor size)-drug concentration curves, mouse weight change curves, and survival curves are plotted. Transgenic B-huTIGIT mice bearing CT26 colon cancer cells are randomly divided into three groups: a negative control group (n=7 per group), a positive antibody (PcAb(4.1D3)) control group (n=6 per group), and a humanized antibody group (n=6 per group); each antibody group is administered at 10 mg / kg and 3 mg / kg. After intraperitoneal administration, the results of treatment in the tumor-bearing mice are as follows: Figure 20 As shown: hTIGIT humanized antibodies h60H5, h51C1 and positive control PcAb (4.1D3) can all effectively slow down tumor growth rate. Figure 20 In the experiment (A), there were no significant differences in body weight changes among the groups of mice. Figure 20 (B) Mice that reached the experimental endpoint were euthanized by cervical dislocation. The survival curves of the mice were plotted using Prism 5.0 software, and the results are as follows: Figure 21 As shown in the treatment-survival curves, both the high and low dose groups of the hTIGI T humanized antibody h51C1 and the high dose group of h60H5 can effectively improve the survival rate of mice.

[0191] Example 18: Tumor Re-challenge Experiment in Tumor-Bearing Healed Mice

[0192] During the efficacy experiment of humanized hTIGIT antibody against tumor-bearing transgenic mice in Example 17, it was found that the positive control PcAb (4.1D3) at 10 mg / kg, the humanized TIGIT antibody h51C1 at 3 mg / kg, and h60H5 at 10 mg / kg all showed tumor regression or even disappearance. Considering that TIGIT antibody has the function of inducing immune memory in the body, in order to verify this hypothesis, a design was made to re-inoculate the tumor-regressed mice (three groups, one mouse in each group) with the colon cancer cell line CT26.WT. The method of re-inoculating CT26 cells was the same as that described in Example 17. At the same time, a blank control group (3 mice in total) was set up with 1×10 6 100 μL of cells per mouse was administered via subcutaneous injection into the axilla (on the side not previously inoculated with tumor cells), followed by treatment according to the prescribed dosage and frequency. Tumor growth and weight changes in the mice were observed and recorded. Experimental results are as follows: Figure 22 As shown: From day 8 to day 25 after CT26.WT injection, the tumors in the blank control group continued to grow until they exceeded 2000 mm. 3 The tumors in the antibody treatment group stopped growing. Figure 22 (A) and there was no significant difference in body weight among the groups of mice. Figure 22 (B)

[0193] Example 19: Efficacy experiment of hTIGIT humanized antibody against 4T1 breast cancer cell line tumor-bearing mice

[0194] The method for inducing B-huTIGIT transgenic mice to bear tumors using 4T1 breast cancer cells was the same as in Example 17. After randomization, the B-huTIGIT transgenic mice were divided into three groups: a negative control group (n=5), a positive antibody control group (n=6) at 10 mg / kg, and humanized antibody groups (h51C and h60H5, 10 mg / kg, n=6 per group). After intraperitoneal treatment, the results of the tumor-bearing mice are as follows... Figure 23 As shown: the tumor volume in the positive control group PcAb (4.1D3) continued to increase, with no difference compared to the blank control group; the tumor volume in the humanized antibody h51C1 group showed a decreasing trend from day 13; the h60H5 group showed a significant reduction in tumor volume (size) on day 20. The antibody treatment experiment reached its endpoint on day 21. After the mice were sacrificed, tumor tissue was isolated. The endpoint tumor volume showed that the h60H5 antibody treatment group had the best effect in inhibiting 4T1 tumor proliferation. Figure 23(B, P<0.05). In summary, both TIGIT humanized antibodies h51C1 and h60H5 can slow tumor growth rate. There were no significant differences in mouse body weight among the groups during the experiment. Figure 23 (C). Each hTIGIT antibody significantly prolonged the survival time of 4T1 tumor-bearing mice. Figure 24 ).

[0195] Example 20: Pharmacokinetics of hTIGIT Humanized Antibody

[0196] Adult Balb / c mice (male and female not specified) aged 8–12 weeks were used for in vivo antibody metabolism studies. After purchase, mice were observed and housed in an SPF-grade animal facility, acclimatized for one week, under constant temperature (23±2)℃, humidity 40%–60%RH, and artificial light with 12 hours of illumination and 12 hours of darkness. Co60-irradiated feed and tap water were provided on-site. Mice were weighed and blood was collected (D0) before administration. Mice were grouped according to weight, and the dosage volume was calculated. A positive control group (PcAb(4.1D3)) and test drug experimental groups (TIGIT humanized antibodies h51C1 and h60H5) were set up according to experimental requirements (n=5 per group). Intravenous administration was performed according to the dosage relationship between mouse weight and dosage (10 mg / kg (mpk) and 3 mg / kg (mpk) for each antibody group). Blood samples of 100 μL per mouse were collected via the retro-orbital venous plexus at 30 min, 1 h, 2 h, 4 h, 24 h, 48 h, 4 d, 8 d, and 11 d after drug administration. The blood was allowed to stand at room temperature for 30 min, then centrifuged at 4000 rpm for 10 min, and serum was collected. Blood collection was terminated when the drug concentration at the last collection point was 1 / 20 of the highest drug concentration. Blood drug concentration was detected using ELISA. The metabolism of the positive control group and humanized antibodies in mice was also investigated. Specifically, 0.5 μg / mL TIGIT-his antigen was coated with the test serum diluent, and the absorbance was detected by colorimetric analysis. Four-parameter curves of the positive control (PcAb(4.1D3)) and candidate humanized antibodies (h51C1 and h60H5) were obtained using data processing software, and the EC50 was calculated. The concentration (Cmax), half-life (T1 / 2), and area under the curve (AUC) at each time point were also calculated for each group. The metabolic curves of the antibody drugs showed that each hTIGIT antibody had relatively good pharmacokinetic activity in mice, with no sudden disappearance of antibody concentration and a relatively gradual decrease in blood concentration. Figure 25The main pharmacokinetic parameters of the positive control 4.1D3, humanized antibodies h51C1, and h60H5 were calculated using the data processing software PK Solution 2.0. These parameters included the area under the plasma concentration-time curve (AUC(0-t)), AUC(0-∞), peak concentration (Cmax), time to peak concentration (Tmax), elimination half-life (T1 / 2), apparent volume of distribution (Vd), and clearance rate (CL). The experimental results are shown in Table 2. The h51C1 and h60H5 drug concentrations exhibited an initial concentration-dependent change in plasma concentration, and the plasma antibody concentration decreased to 1 / 20 or less of the highest value after day 11.

[0197] Table 2. Mean values ​​of major pharmacokinetic parameters (Mean ± SD) for each dosage group of TIGIT humanized antibody.

[0198]

[0199] Example 21: Pharmacodynamic experiment of hTIGIT humanized antibody combined with PD-L1 antibody drug in treating CT26 cell line tumor-bearing mice with colon cancer.

[0200] Antibody efficacy studies were conducted using 8-12 week old adult Balb / c-huTIGIT (B-huTIGIT) transgenic female mice (purchased from Nanjing Jicui Pharmaceutical Co., Ltd.). Animal husbandry and CT26 cell xenograft methods were the same as in Example 17. After randomizing B-huTIGIT transgenic mice bearing CT26 colon cancer cells, the following groups were established: a negative control group (PBS group; 6 mice); an atezolizumab group (10 mg / kg / mpk, purchased from Roche, batch number H0217B01; 5 mice); a humanized antibody h60H5- group (10 mg / kg / mpk, 6 mice); an atezolizumab (10 mg / kg) combined with h51C1 group (h51C1 concentrations of 10 mg / kg and 3 mg / kg / mpk); and an atezolizumab (10 mg / kg) combined with h60H5 group (h60H5 concentration of 10 mg / kg / mpk and atezolizumab dose of 10 mg / kg / mpk). The results of intraperitoneal treatment in the tumor-bearing mice are as follows: Figure 26 As shown: Both the atezolizumab group and the atezolizumab combined with h51C1 group could inhibit tumor growth. In particular, the atezolizumab combined with h51C1 group showed better tumor growth inhibition than the atezolizumab group. Figure 26(A); Atezolizumab group, humanized antibody h60H5 group, and atezolizumab combined with h60H5 group all inhibited tumor growth. Among them, the humanized antibody h60H5 group and the atezolizumab combined with h60H5 group were more effective in inhibiting tumor growth than the atezolizumab group, and the atezolizumab combined with h60H5 group was more effective than the humanized antibody h60H5 group. Figure 26 As can be seen from Figure B, the combination of h51C1 / h60H5 provided by this invention with PD-L1 antibody (atezolizumab) has better technical effects in the treatment of tumors.

[0201] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Human TIGIT antibody or its antigen-binding fragment, 60H5; The 60H5 includes a 60H5 heavy chain variable region and a 60H5 light chain variable region; The 60H5 heavy chain variable region includes CDR1, CDR2, and CDR3; The amino acid sequence of the CDR1 variable region of the 60H5 heavy chain is: GYTFTEYT (SEQ ID NO.2). The amino acid sequence of the CDR2 variable region of the 60H5 heavy chain is: INPNGGT (SEQ ID NO.3); The amino acid sequence of the CDR3 variable region of the 60H5 heavy chain is: ARSGNWDYAMDY (SEQ ID NO.4). The variable region of the 60H5 light chain includes CDR1, CDR2, and CDR3; The amino acid sequence of the CDR1 variable region of the 60H5 light chain is: QHVSTA (SEQ ID NO.7); The amino acid sequence of the CDR2 variable region of the 60H5 light chain is: SAS (SEQ ID NO.8); The amino acid sequence of the CDR3 variable region of the 60H5 light chain is: QQHYITPWT (SEQ ID NO.9).

2. The human TIGIT antibody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the 60H5 heavy chain variable region includes: OR QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGQRLEWIGGINPNNGGTSYNQKFQGRVTITVDTSASTAYMELSSLRSEDTAVYYCARSGNWDYAMDYWGQGTTVTVSS (SEQ ID NO. 21); The amino acid sequence of the 60H5 light chain variable region includes: OR DIQMTQSPSSMSASVGDRVTITCKASQHVSTAVVWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYITPWTFGGGTKLEIKRTVA (SEQ ID NO. 22).

3. A nucleic acid molecule encoding the human TIGIT antibody or its antigen-binding fragment as described in claim 1 or 2.

4. An expression cassette, recombinant vector, or transgenic cell line containing the nucleic acid molecule of claim 3.

5. The use of the human TIGIT antibody or its antigen-binding fragment as described in claim 1 or 2 in the preparation of products for detecting TIGIT or in the preparation of medicaments for treating tumors; The tumors were selected from colon cancer and breast cancer.

6. The application according to claim 5, characterized in that: The product contains at least one of reagents, test plates, and kits.

7. A product comprising: the human TIGIT antibody of claim 1 or 2 or an antigen-binding fragment thereof; The product contains at least one of reagents, test plates, and kits.

8. A pharmaceutical composition comprising the human TIGIT antibody of claim 1 or 2 or an antigen-binding fragment thereof.

9. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition also contains other antitumor drugs; The other anti-tumor drugs mentioned are: PD-L1 antibodies.

10. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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