TIGIT Antibodies and Their Uses

Abstract

The present invention provides an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, a composition comprising the anti-TIGIT antibody or an antigen-binding fragment thereof, and uses thereof in the diagnosis and treatment of TIGIT-related diseases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of the PCT application with the application number PCT / CN2021 / 139122, titled "TIGIT Antibody and Its Uses", filed on December 17, 2021, the disclosure of which is incorporated herein by reference. Technical field

[0003] The present invention relates to an antibody or an antigen - binding fragment thereof that specifically binds to TIGIT (T - cell immunoreceptor with Ig and ITIM domains), a pharmaceutical composition comprising the anti - TIGIT antibody or its antigen - binding fragment, and their uses. Background art

[0004] In recent years, tumor immunotherapy has made great breakthroughs and become a new hope for tumor therapy. In particular, treatment regimens that block tumor immune - inhibitory checkpoints represented by PD - 1 / PD - L1 and CTLA - 4 have attracted much attention. Since 2000, the FDA has successively approved PD - 1 / PD - L1 monoclonal antibodies for clinical treatment of malignant tumors such as melanoma, non - small cell lung cancer, and prostate cancer, and achieved good therapeutic effects. However, the clinical response rate of PD - 1 / PD - L1 monoclonal antibody treatment is still limited, which greatly restricts its clinical application. Therefore, searching for new immune - inhibitory checkpoints has become a research hotspot.

[0005] TIGIT is a new immune - inhibitory factor discovered by the Genentech team in 2009 (Nat Immunol, 2009, 10: 48 - 57). It is a member of the PVR - like protein family. TIGIT is expressed in T cells and NK cells, and these cells include CD4 + T cells, CD8 +T cells and Treg cells. Under normal circumstances, the expression level of TIGIT is low, but when T cells and NK cells are activated, the expression of TIGIT increases significantly (J Immunol, 2012, 188: 3869-3875, Cancer Cell 26, 923–937, Nat Immunol, 19, 723-732). Currently, the discovered TIGIT ligands include CD155, CD112, and CD113, among which the main ligand of TIGIT is CD155. Crystal structure analysis shows that TIGIT and CD155 form homodimers respectively, and they further form a heterotetramer through the interaction between the ligand and the receptor (Proc Natl Acad Sci USA 2012; 109: 5399–404). The binding affinity of TIGIT to CD112 or CD113 is significantly lower than that to CD155. CD155 is mainly expressed in dendritic cells, T cells, B cells, macrophages, and non-hematopoietic tissues (such as the kidney, nervous system, and small intestine). Similar to TIGIT, the activating receptors DNAM-1 and CD96 can also bind to CD155, but their affinity is weaker than that of TIGIT. In summary, the ligand-receptor interaction mode of TIGIT / CD155 is similar to the CTLA-4 / CD28 pathway. The inhibitory receptor with high affinity and the activating receptor with low affinity compete to bind the same ligand, thereby precisely regulating the immune response. TIGIT binding to CD155 can exert immunosuppressive effects by regulating DC function, inhibiting the activity of effector T cells, interfering with DNAM-1 co-stimulation, and increasing Treg suppression (Clinical and Experimental Immunology, 2020 May; 200(2): 108-119, Immunity 40, 569–581).

[0006] Several human and mouse studies have shown that TIGIT is highly expressed in tumor-infiltrating lymphocytes. TIGIT is upregulated in many malignant tumors, including melanoma, breast cancer, non-small cell lung cancer, colorectal adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma (Clinical and Experimental Immunology, 2020 May; 200(2): 108-119). Some studies have also found that TIGIT is highly expressed in CD8 + T cells, tumor-infiltrating Tregs, and NK cells. In tumor patients, tumor-infiltrating CD8 +The expression of TIGIT in T cells and NK cells is generally consistent with the high expression of other inhibitory receptors (such as PD-1, LAG-3, Tim-3) and the low expression of DNAM-1. The high expression of TIGIT is often associated with poor prognosis of malignant tumors. The high expression of TIGIT in NK cells is related to the severity of the disease. The tumor growth of TIGIT knockout mice is significantly reduced and the survival rate increases.

[0007] Due to its macromolecular properties, antibody drugs are usually accompanied by immune-related adverse events (irAE). In TIGIT knockout mice, there are no spontaneous autoimmune symptoms and no hematopoietic cell developmental disorders. The incidence of autoimmune diseases only increases after hybridization with mice with a tendency to autoimmune diseases. Compared with PD-1 and CTLA-4 mAb, animal experiments show that the incidence of irAE is lower during the administration of anti-TIGIT mAb (Oncoimmunology 2018; 7: e1445949). Therefore, antibody drugs targeting TIGIT have a relatively low risk of side effects and it is a high-quality candidate target for clinical anti-cancer drugs.

[0008] The published clinical trial results show that the combination of TIGIT antibody and PD-1 / PD-L1 monoclonal antibody can significantly improve the response rate of patients, improve the treatment effect, and solve the drug resistance of some patients (Cancers 2019; 11: 877, Cancer Discov, 10: 1086 - 1087 (2020)). Currently, no monoclonal antibody targeting TIGIT has been approved for marketing globally. Therefore, it is necessary to develop antibodies with high affinity and activity as candidate drugs. Summary of the Invention

[0009] After a large number of experiments, the inventors of the present invention used a transgenic chromosome mouse (TC mAb TM mouse) platform to screen and unexpectedly obtained an antibody that specifically binds to TIGIT. The antibody shows excellent affinity for TIGIT and has potential for drug development.

[0010] The present invention provides improved drugs and treatment methods for cancer and chronic viral infections, which comprise anti-TIGIT antibodies or antigen-binding fragments thereof that specifically bind to human TIGIT (huTIGIT). Isolated antibodies are provided herein, such as monoclonal antibodies, particularly human monoclonal antibodies, which specifically bind to huTIGIT and have desired functional properties, such as high-affinity specific binding to huTIGIT, binding to simian TIGIT (e.g., cynomolgus TIGIT), the ability to block the binding of TIGIT to PVR and nectin-2, the ability to block the interaction of TIGIT with DNAM, or any combination of these properties.

[0011] The present invention relates to antibodies that compete with antibodies having the heavy and light chain variable domain sequences disclosed herein for binding to huTIGIT and cross-block the binding of antibodies having the heavy and light chain variable domain sequences disclosed herein to huTIGIT.

[0012] In certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention enhance anti-tumor immune responses, e.g., antigen-specific T cell responses. In other embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention block TIGIT-mediated inhibitory signaling, allowing PVR / DNAM co-stimulation of NK cells to increase NK-mediated anti-tumor response killing. In another embodiment, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention deplete the regulatory T cell population within the tumor, which would otherwise inhibit the anti-tumor immune response. In another embodiment, the anti-TIGIT antibodies of the present invention in the form of IgG1 deplete CD8+ exhausted T cells and Tregs, thereby allowing the influx of fresh non-exhausted CD8+ T cells. In some embodiments, the anti-TIGIT antibodies of the present invention in the form of IgG1 increase the proportion of the CD8+ TIL population in the tumor microenvironment. In other embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention act through one or more of the above mechanisms, as the mechanisms are not necessarily mutually exclusive.

[0013] In certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention do not bind to activating Fcγ receptors (FcγR), e.g., in embodiments where the anti-tumor activity of TIGIT-expressing cells is enhanced. In alternative embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention bind to one or more activating FcγR, e.g., in embodiments where the killing of TIGIT-expressing cells (such as exhausted CD8+ T cells or Tregs) is dependent.

[0014] In a first aspect, the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to TIGIT. The anti-TIGIT antibody or antigen-binding fragment comprises a heavy-chain variable region (VH) and / or a light-chain variable region (VL), and wherein the heavy-chain variable region comprises CDRH1, CDRH2, and CDRH3, and the light-chain variable region comprises CDRL1, CDRL2, and CDRL3.

[0015] In some embodiments of the present invention, wherein

[0016] (a) the CDRH1 comprises the sequence of SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 65, SEQ ID NO: 75, or SEQ ID NO: 81; or the CDRH1 comprises a sequence derived from SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 65, SEQ ID NO: 75, or SEQ ID NO: 81 by addition, deletion, or substitution of one or more amino acids;

[0017] (b) the CDRH2 comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 76, or SEQ ID NO: 82; or the CDRH1 comprises a sequence derived from SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 76, or SEQ ID NO: 82 by addition, deletion, or substitution of one or more amino acids; and

[0018] (c) The CDRH3 comprises the sequence of SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:77 or SEQ ID NO:83; or the CDRH1 comprises a sequence derived from SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:77 or SEQ ID NO:83 by addition, deletion or substitution of one or more amino acids.

[0019] In some other embodiments of the present invention, wherein

[0020] (a) The CDRL1 comprises the sequence of SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:72 or SEQ ID NO:78; or the CDRH1 comprises a sequence derived from SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:72 or SEQ ID NO:78 by addition, deletion or substitution of one or more amino acids;

[0021] (b) The CDRL2 comprises the sequence of SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:73 or SEQ ID NO:79; or the CDRH1 comprises a sequence derived from SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:73 or SEQ ID NO:79 by addition, deletion or substitution of one or more amino acids; and

[0022] (c) The CDRL3 comprises the sequence of SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:74 or SEQ ID NO:80; or the CDRH1 comprises a sequence derived from SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:74 or SEQ ID NO:80 by addition, deletion or substitution of one or more amino acids.

[0023] In some other embodiments of the present invention, wherein

[0024] (a) The CDRH1 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:75 and SEQ ID NO:81;

[0025] (b) The CDRH2 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:76 and SEQ ID NO:82; and

[0026] (c) The CDRH3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:77 and SEQ ID NO:83; and / or

[0027] (d) The CDRL1 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:72 and SEQ ID NO:78;

[0028] (e) The CDRL2 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:73 and SEQ ID NO:79; and

[0029] (f) The CDRL3 comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to a sequence selected from the group consisting of: SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:74, and SEQ ID NO:80.

[0030] In some other embodiments of the present invention, wherein

[0031] (a) The CDRH1 comprises a sequence selected from the group consisting of: SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:75, and SEQ ID NO:81;

[0032] (b) The CDRH2 comprises a sequence selected from the group consisting of: SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:76, and SEQ ID NO:82;

[0033] (c) The CDRH3 comprises a sequence selected from the group consisting of: SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:77, and SEQ ID NO:83;

[0034] (d) The CDRL1 comprises a sequence selected from the group consisting of: SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:72, and SEQ ID NO:78;

[0035] (e) The CDRL2 comprises a sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO:73, and SEQ ID NO:79; and

[0036] (f) The CDRL3 comprises a sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:74, and SEQ ID NO:80.

[0037] In some other embodiments of the present invention, wherein the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3, and wherein

[0038] (a) The CDRH1 comprises the sequence shown in SEQ ID NO:23, the CDRH2 comprises the sequence shown in SEQ ID NO:24, and the CDRH3 comprises the sequence shown in SEQ ID NO:25;

[0039] (b) The CDRH1 comprises the sequence shown in SEQ ID NO:29, the CDRH2 comprises the sequence shown in SEQ ID NO:30, and the CDRH3 comprises the sequence shown in SEQ ID NO:31;

[0040] (c) The CDRH1 comprises the sequence shown in SEQ ID NO:35, the CDRH2 comprises the sequence shown in SEQ ID NO:36, and the CDRH3 comprises the sequence shown in SEQ ID NO:37;

[0041] (d) The CDRH1 comprises the sequence shown in SEQ ID NO:41, the CDRH2 comprises the sequence shown in SEQ ID NO:42, and the CDRH3 comprises the sequence shown in SEQ ID NO:43;

[0042] (e) The CDRH1 comprises the sequence shown in SEQ ID NO: 47, the CDRH2 comprises the sequence shown in SEQ ID NO: 48, and the CDRH3 comprises the sequence shown in SEQ ID NO: 49;

[0043] (f) The CDRH1 comprises the sequence shown in SEQ ID NO: 53, the CDRH2 comprises the sequence shown in SEQ ID NO: 54, and the CDRH3 comprises the sequence shown in SEQ ID NO: 55;

[0044] (g) The CDRH1 comprises the sequence shown in SEQ ID NO: 59, the CDRH2 comprises the sequence shown in SEQ ID NO: 60, and the CDRH3 comprises the sequence shown in SEQ ID NO: 61;

[0045] (h) The CDRH1 comprises the sequence shown in SEQ ID NO: 65, the CDRH2 comprises the sequence shown in SEQ ID NO: 66, and the CDRH3 comprises the sequence shown in SEQ ID NO: 67;

[0046] (i) The CDRH1 comprises the sequence of SEQ ID NO: 75, the CDRH2 comprises the sequence of SEQ ID NO: 76, and the CDRH3 comprises the sequence of SEQ ID NO: 77; or

[0047] (j) The CDRH1 comprises the sequence of SEQ ID NO: 81, the CDRH2 comprises the sequence of SEQ ID NO: 82, and the CDRH3 comprises the sequence of SEQ ID NO: 83.

[0048] In some other embodiments of the present invention, wherein the light chain variable region comprises CDRL1, CDRL2, and CDRL3, and wherein

[0049] (a) The CDRL1 comprises the sequence shown in SEQ ID NO: 20, the CDRL2 comprises the sequence shown in SEQ ID NO: 21, and the CDRL3 comprises the sequence shown in SEQ ID NO: 22;

[0050] (b) The CDRL1 comprises the sequence shown in SEQ ID NO: 26, the CDRL2 comprises the sequence shown in SEQ ID NO: 27, and the CDRL3 comprises the sequence shown in SEQ ID NO: 28;

[0051] (c) The CDRL1 comprises the sequence shown in SEQ ID NO:32, the CDRL2 comprises the sequence shown in SEQ ID NO:33, and the CDRL3 comprises the sequence shown in SEQ ID NO:34;

[0052] (d) The CDRL1 comprises the sequence shown in SEQ ID NO:38, the CDRL2 comprises the sequence shown in SEQ ID NO:39, and the CDRL3 comprises the sequence shown in SEQ ID NO:40;

[0053] (e) The CDRL1 comprises the sequence shown in SEQ ID NO:44, the CDRL2 comprises the sequence shown in SEQ ID NO:45, and the CDRL3 comprises the sequence shown in SEQ ID NO:46;

[0054] (f) The CDRL1 comprises the sequence shown in SEQ ID NO:50, the CDRL2 comprises the sequence shown in SEQ ID NO:51, and the CDRL3 comprises the sequence shown in SEQ ID NO:52;

[0055] (g) The CDRL1 comprises the sequence shown in SEQ ID NO:56, the CDRL2 comprises the sequence shown in SEQ ID NO:57, and the CDRL3 comprises the sequence shown in SEQ ID NO:58;

[0056] (h) The CDRL1 comprises the sequence shown in SEQ ID NO:62, the CDRL2 comprises the sequence shown in SEQ ID NO:63, and the CDRL3 comprises the sequence shown in SEQ ID NO:64;

[0057] (i) The CDRL1 comprises the sequence of SEQ ID NO:72, the CDRL2 comprises the sequence of SEQ ID NO:73, and the CDRL3 comprises the sequence of SEQ ID NO:74; or

[0058] (j) The CDRL1 comprises the sequence of SEQ ID NO:78, the CDRL2 comprises the sequence of SEQ ID NO:79, and the CDRL3 comprises the sequence of SEQ ID NO:80.

[0059] In some other embodiments of the present invention, wherein

[0060] (a) The CDRH1 comprises the sequence shown in SEQ ID NO:23, the CDRH2 comprises the sequence shown in SEQ ID NO:24, and the CDRH3 comprises the sequence shown in SEQ ID NO:25; and the CDRL1 comprises the sequence shown in SEQ ID NO:20, the CDRL2 comprises the sequence shown in SEQ ID NO:21, and the CDRL3 comprises the sequence shown in SEQ ID NO:22;

[0061] (b) The CDRH1 comprises the sequence shown in SEQ ID NO:29, the CDRH2 comprises the sequence shown in SEQ ID NO:30, and the CDRH3 comprises the sequence shown in SEQ ID NO:31; and the CDRL1 comprises the sequence shown in SEQ ID NO:26, the CDRL2 comprises the sequence shown in SEQ ID NO:27, and the CDRL3 comprises the sequence shown in SEQ ID NO:28;

[0062] (c) The CDRH1 comprises the sequence shown in SEQ ID NO:35, the CDRH2 comprises the sequence shown in SEQ ID NO:36, and the CDRH3 comprises the sequence shown in SEQ ID NO:37; and the CDRL1 comprises the sequence shown in SEQ ID NO:32, the CDRL2 comprises the sequence shown in SEQ ID NO:33, and the CDRL3 comprises the sequence shown in SEQ ID NO:34;

[0063] (d) The CDRH1 comprises the sequence shown in SEQ ID NO:41, the CDRH2 comprises the sequence shown in SEQ ID NO:42, and the CDRH3 comprises the sequence shown in SEQ ID NO:43; and the CDRL1 comprises the sequence shown in SEQ ID NO:38, the CDRL2 comprises the sequence shown in SEQ ID NO:39, and the CDRL3 comprises the sequence shown in SEQ ID NO:40;

[0064] (e) The CDRH1 comprises the sequence shown in SEQ ID NO:47, the CDRH2 comprises the sequence shown in SEQ ID NO:48, and the CDRH3 comprises the sequence shown in SEQ ID NO:49; and the CDRL1 comprises the sequence shown in SEQ ID NO:44, the CDRL2 comprises the sequence shown in SEQ ID NO:45, and the CDRL3 comprises the sequence shown in SEQ ID NO:46;

[0065] (f) The CDRH1 comprises the sequence shown in SEQ ID NO:53, the CDRH2 comprises the sequence shown in SEQ ID NO:54, and the CDRH3 comprises the sequence shown in SEQ ID NO:55; and the CDRL1 comprises the sequence shown in SEQ ID NO:50, the CDRL2 comprises the sequence shown in SEQ ID NO:51, and the CDRL3 comprises the sequence shown in SEQ ID NO:52;

[0066] (g) The CDRH1 comprises the sequence shown in SEQ ID NO:59, the CDRH2 comprises the sequence shown in SEQ ID NO:60, and the CDRH3 comprises the sequence shown in SEQ ID NO:61; and the CDRL1 comprises the sequence shown in SEQ ID NO:56, the CDRL2 comprises the sequence shown in SEQ ID NO:57, and the CDRL3 comprises the sequence shown in SEQ ID NO:58;

[0067] (h) The CDRH1 comprises the sequence shown in SEQ ID NO:65, the CDRH2 comprises the sequence shown in SEQ ID NO:66, and the CDRH3 comprises the sequence shown in SEQ ID NO:67; and the CDRL1 comprises the sequence shown in SEQ ID NO:62, the CDRL2 comprises the sequence shown in SEQ ID NO:63, and the CDRL3 comprises the sequence shown in SEQ ID NO:64;

[0068] (i) The CDRH1 comprises the sequence of SEQ ID NO:75, the CDRH2 comprises the sequence of SEQ ID NO:76, and the CDRH3 comprises the sequence of SEQ ID NO:77; and the CDRL1 comprises the sequence of SEQ ID NO:72, the CDRL2 comprises the sequence of SEQ ID NO:73, and the CDRL3 comprises the sequence of SEQ ID NO:74; or

[0069] (j) The CDRH1 comprises the sequence of SEQ ID NO:81, the CDRH2 comprises the sequence of SEQ ID NO:82, and the CDRH3 comprises the sequence of SEQ ID NO:83; and the CDRL1 comprises the sequence of SEQ ID NO:78, the CDRL2 comprises the sequence of SEQ ID NO:79, and the CDRL3 comprises the sequence of SEQ ID NO:80.

[0070] In some other embodiments of the present invention, wherein

[0071] (a) The sequence of CDRH1 is as shown in SEQ ID NO:23, the sequence of CDRH2 is as shown in SEQ ID NO:24, and the sequence of CDRH3 is as shown in SEQ ID NO:25; and the sequence of CDRL1 is as shown in SEQ ID NO:20, the sequence of CDRL2 is as shown in SEQ ID NO:21, and the sequence of CDRL3 is as shown in SEQ ID NO:22;

[0072] (b) The sequence of CDRH1 is as shown in SEQ ID NO:29, the sequence of CDRH2 is as shown in SEQ ID NO:30, and the sequence of CDRH3 is as shown in SEQ ID NO:31; and the sequence of CDRL1 is as shown in SEQ ID NO:26, the sequence of CDRL2 is as shown in SEQ ID NO:27, and the sequence of CDRL3 is as shown in SEQ ID NO:28;

[0073] (c) The sequence of CDRH1 is as shown in SEQ ID NO:35, the sequence of CDRH2 is as shown in SEQ ID NO:36, and the sequence of CDRH3 is as shown in SEQ ID NO:37; and the sequence of CDRL1 is as shown in SEQ ID NO:32, the sequence of CDRL2 is as shown in SEQ ID NO:33, and the sequence of CDRL3 is as shown in SEQ ID NO:34;

[0074] (d) The sequence of CDRH1 is as shown in SEQ ID NO: 41, the sequence of CDRH2 is as shown in SEQ ID NO: 42, and the sequence of CDRH3 is as shown in SEQ ID NO: 43; and the sequence of CDRL1 is as shown in SEQ ID NO: 38, the sequence of CDRL2 is as shown in SEQ ID NO: 39, and the sequence of CDRL3 is as shown in SEQ ID NO: 40;

[0075] (e) The sequence of CDRH1 is as shown in SEQ ID NO: 47, the sequence of CDRH2 is as shown in SEQ ID NO: 48, and the sequence of CDRH3 is as shown in SEQ ID NO: 49; and the sequence of CDRL1 is as shown in SEQ ID NO: 44, the sequence of CDRL2 is as shown in SEQ ID NO: 45, and the sequence of CDRL3 is as shown in SEQ ID NO: 46;

[0076] (f) The sequence of CDRH1 is as shown in SEQ ID NO: 53, the sequence of CDRH2 is as shown in SEQ ID NO: 54, and the sequence of CDRH3 is as shown in SEQ ID NO: 55; and the sequence of CDRL1 is as shown in SEQ ID NO: 50, the sequence of CDRL2 is as shown in SEQ ID NO: 51, and the sequence of CDRL3 is as shown in SEQ ID NO: 52;

[0077] (g) The sequence of CDRH1 is as shown in SEQ ID NO: 59, the sequence of CDRH2 is as shown in SEQ ID NO: 60, and the sequence of CDRH3 is as shown in SEQ ID NO: 61; and the sequence of CDRL1 is as shown in SEQ ID NO: 56, the sequence of CDRL2 is as shown in SEQ ID NO: 57, and the sequence of CDRL3 is as shown in SEQ ID NO: 58;

[0078] (h) The sequence of CDRH1 is as shown in SEQ ID NO: 65, the sequence of CDRH2 is as shown in SEQ ID NO: 66, and the sequence of CDRH3 is as shown in SEQ ID NO: 67; and the sequence of CDRL1 is as shown in SEQ ID NO: 62, the sequence of CDRL2 is as shown in SEQ ID NO: 63, and the sequence of CDRL3 is as shown in SEQ ID NO: 64;

[0079] (i) The sequence of CDRH1 is as shown in SEQ ID NO:75, the sequence of CDRH2 is as shown in SEQ ID NO:76, and the sequence of CDRH3 is as shown in SEQ ID NO:77; and the sequence of CDRL1 is as shown in SEQ ID NO:72, the sequence of CDRL2 is as shown in SEQ ID NO:73, and the sequence of CDRL3 is as shown in SEQ ID NO:74; or

[0080] (j) The sequence of CDRH1 is as shown in SEQ ID NO:81, the sequence of CDRH2 is as shown in SEQ ID NO:82, and the sequence of CDRH3 is as shown in SEQ ID NO:83; and the sequence of CDRL1 is as shown in SEQ ID NO:78, the sequence of CDRL2 is as shown in SEQ ID NO:79, and the sequence of CDRL3 is as shown in SEQ ID NO:80.

[0081] In some other embodiments of the present invention, wherein the heavy chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:69 and SEQ ID NO:71.

[0082] In some other embodiments of the present invention, wherein the light chain variable region comprises a sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:68 and SEQ ID NO:70.

[0083] In some other embodiments of the present invention, wherein

[0084] (a) The heavy chain variable region comprises the sequence shown in SEQ ID NO:5, and the light chain variable region comprises the sequence shown in SEQ ID NO:4;

[0085] (b) The heavy chain variable region comprises the sequence shown in SEQ ID NO:7, and the light chain variable region comprises the sequence shown in SEQ ID NO:6;

[0086] (c) The heavy chain variable region contains the sequence shown in SEQ ID NO:9, and the light chain variable region contains the sequence shown in SEQ ID NO:8;

[0087] (d) The heavy chain variable region contains the sequence shown in SEQ ID NO:11, and the light chain variable region contains the sequence shown in SEQ ID NO:10;

[0088] (e) The heavy chain variable region contains the sequence shown in SEQ ID NO:13, and the light chain variable region contains the sequence shown in SEQ ID NO:12;

[0089] (f) The heavy chain variable region contains the sequence shown in SEQ ID NO:15, and the light chain variable region contains the sequence shown in SEQ ID NO:14;

[0090] (g) The heavy chain variable region contains the sequence shown in SEQ ID NO:17, and the light chain variable region contains the sequence shown in SEQ ID NO:16; or

[0091] (h) The heavy chain variable region contains the sequence shown in SEQ ID NO:19, and the light chain variable region contains the sequence shown in SEQ ID NO:18.

[0092] In some other embodiments of the present invention, wherein

[0093] (a) The sequence of the heavy chain variable region is as shown in SEQ ID NO:5, and the sequence of the light chain variable region is as shown in SEQ ID NO:4;

[0094] (b) The sequence of the heavy chain variable region is as shown in SEQ ID NO:7, and the sequence of the light chain variable region is as shown in SEQ ID NO:6;

[0095] (c) The sequence of the heavy chain variable region is as shown in SEQ ID NO:9, and the sequence of the light chain variable region is as shown in SEQ ID NO:8;

[0096] (d) The sequence of the heavy chain variable region is as shown in SEQ ID NO:11, and the sequence of the light chain variable region is as shown in SEQ ID NO:10;

[0097] (e) The sequence of the heavy chain variable region is as shown in SEQ ID NO:13, and the sequence of the light chain variable region is as shown in SEQ ID NO:12;

[0098] (f) The sequence of the heavy chain variable region is as shown in SEQ ID NO: 15, and the sequence of the light chain variable region is as shown in SEQ ID NO: 14;

[0099] (g) The sequence of the heavy chain variable region is as shown in SEQ ID NO: 17, and the sequence of the light chain variable region is as shown in SEQ ID NO: 16;

[0100] (h) The sequence of the heavy chain variable region is as shown in SEQ ID NO: 19, and the sequence of the light chain variable region is as shown in SEQ ID NO: 18;

[0101] (i) The heavy chain variable region contains the sequence of SEQ ID NO: 69, and the light chain variable region contains the sequence of SEQ ID NO: 68; or

[0102] (j) The heavy chain variable region contains the sequence of SEQ ID NO: 71, and the light chain variable region contains the sequence of SEQ ID NO: 70.

[0103] In some other embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and wherein

[0104] (a) The VH comprises CDRH1, CDRH2, and CDRH3 of VH as shown in SEQ ID NO: 5, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of VL as shown in SEQ ID NO: 4;

[0105] (b) The VH comprises CDRH1, CDRH2, and CDRH3 of VH as shown in SEQ ID NO: 7, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of VL as shown in SEQ ID NO: 6;

[0106] (c) The VH comprises CDRH1, CDRH2, and CDRH3 of VH as shown in SEQ ID NO: 9, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of VL as shown in SEQ ID NO: 8;

[0107] (d) The VH comprises CDRH1, CDRH2, and CDRH3 of VH as shown in SEQ ID NO: 11, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of VL as shown in SEQ ID NO: 10;

[0108] (e) The VH comprises CDRH1, CDRH2 and CDRH3 of VH as shown in SEQ ID NO:13, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of VL as shown in SEQ ID NO:12;

[0109] (f) The VH comprises CDRH1, CDRH2 and CDRH3 of VH as shown in SEQ ID NO:15, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of VL as shown in SEQ ID NO:14;

[0110] (g) The VH comprises CDRH1, CDRH2 and CDRH3 of VH as shown in SEQ ID NO:17, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of VL as shown in SEQ ID NO:16;

[0111] (h) The VH comprises CDRH1, CDRH2 and CDRH3 of VH as shown in SEQ ID NO:19, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of VL as shown in SEQ ID NO:18;

[0112] (i) The VH comprises CDRH1, CDRH2 and CDRH3 of VH as shown in SEQ ID NO:69, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of VL as shown in SEQ ID NO:68; or

[0113] (j) The VH comprises CDRH1, CDRH2 and CDRH3 of VH as shown in SEQ ID NO:71, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of VL as shown in SEQ ID NO:70.

[0114] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, (a) some antibodies can block the binding of themselves (1B2-8C) and Tiragolumab, 4A042-H3, 4A042-H7 and 4B030a, partially block the binding of 4A063, but cannot block the binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a; (b) some antibodies can block the binding of themselves (4A042-H3), 1B2-8C, 4A042-H7 and 4B030a, without blocking the binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a; (c) some antibodies can block the binding of themselves (4A042-H7), 1B2-8C, 4A042-H3 and 4B030a, partially block the binding of 4B037a and 4A063, but cannot block the binding of 4B056a, 4D035a and 4E061a; (d) some antibodies can block the binding of themselves (4B030a), partially block the binding of 4B056a and 4A063, and do not block the binding of 4B037a, 4D035a and 4E061a; (e) some antibodies can block the binding of themselves (4B037a, 4A063), 4B056a, 4D035a and 4E061a, and partially block the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7 and 4B030a; (f) some antibodies can block the binding of themselves (4B056a), 4B037a, 4A063, 4D035a and 4E061a, partially block the binding of Tiragolumab, 1B2-8C, 4A042-H3 and 4B030a, but cannot block the binding of 4A042-H7; (g) some antibodies can block the binding of themselves (4D035a, 4E061a), 4B037a, 4B056a, 4A063, without blocking the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7 and 4B030a.

[0115] In some embodiments, the anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention also binds to cynomolgus monkey TIGIT.

[0116] In some other embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a heavy chain constant region selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0117] In some examples of the present invention, the heavy chain constant region is the heavy chain constant region of human IgG or a variant thereof.

[0118] In some other embodiments of the present invention, the anti-TIGIT antibody or its antigen-binding fragment is in a form selected from the group consisting of: F(ab')2, Fab', Fab, Fv, scFv, bispecific antibodies, and combinations thereof.

[0119] The present invention also provides an immunoconjugate comprising the anti-TIGIT antibody described herein linked to a reagent such as a detectable label or a cytotoxic agent.

[0120] In other embodiments, the antigen-binding domain of the antibody of the present invention is present in a bispecific molecule, which further comprises an antigen-binding domain that specifically binds to a different immune regulatory receptor, including but not limited to PD-1, CTLA-4, or LAG3.

[0121] In a second aspect, the present invention provides a polynucleotide encoding an anti-TIGIT antibody or its antigen-binding fragment.

[0122] In a third aspect, the present invention provides an expression vector that expresses the anti-TIGIT antibody or its antigen-binding fragment.

[0123] In a fourth aspect, the present invention provides an engineered cell comprising a vector that expresses the anti-TIGIT antibody or antigen-binding fragment.

[0124] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the anti-TIGIT antibody or its antigen-binding fragment of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, or the cell of the fourth aspect, and a pharmaceutically acceptable carrier. The present invention further provides an antibody-drug conjugate comprising the anti-TIGIT antibody or antigen-binding fragment of the first aspect. Also provided herein is a kit containing the anti-TIGIT antibody or its antigen-binding fragment and instructions for use.

[0125] In a sixth aspect, the present invention provides the use of the anti-TIGIT antibody or its antigen-binding fragment of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, the cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the manufacture of a drug for the treatment of TIGIT-related diseases. Preferably, the TIGIT-related disease is a T cell dysfunction disease; more preferably, the TIGIT-related disease is a tumor, an immune disease, or an infectious disease; more preferably, the cancer is selected from the group consisting of melanoma, breast cancer, non-small cell lung cancer, colorectal adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma. More preferably, the cells of the tumor are CD155-positive or PVR-positive.

[0126] In some embodiments, the present invention provides a method for enhancing antigen-specific T cell responses, the method comprising contacting T cells with an anti-huTIGIT antibody of the present invention or an antigen-binding fragment thereof, such that the antigen-specific T cell response is enhanced, e.g., enhanced by reducing inhibitory signals that would otherwise attenuate the anti-tumor response. In some embodiments, the antigen-specific T cells are tumor antigen-specific effector T cells, such as CD8+ T cells, and, e.g., the enhancement of anti-tumor activity is caused by blocking TIGIT-mediated inhibitory effects. The anti-huTIGIT antibody of the present invention or an antigen-binding fragment thereof can also reduce inhibitory signals in NK cells and thus increase their anti-tumor activity. Without wishing to be bound by theory, the anti-huTIGIT antibody of the present invention increases effector T cell or NK cell function by blocking the binding of TIGIT to PVR, thereby reducing or eliminating inhibitory signals that would otherwise be delivered to the cells. Alternatively or in addition, the anti-TIGIT antibody of the present invention or an antigen-binding fragment thereof can inhibit the interaction between TIGIT and DNAM-1 / CD226, which would otherwise reduce DNAM-1-mediated immune activation.

[0127] The present invention provides a method for reducing or depleting T regs in tumors in a subject in need thereof, the method comprising administering an effective amount of an anti-huTIGIT antibody of the present invention, wherein the antibody has effector function or enhanced effector function, to reduce the number of T regs in the tumor.

[0128] The present invention provides a method for enhancing an immune response in a subject, the method comprising administering an effective amount of an anti-huTIGIT antibody of the present invention or an antigen-binding fragment thereof to the subject, such that the immune response in the subject is enhanced. In certain embodiments, the subject has a tumor and the immune response against the tumor is enhanced. In another embodiment, the subject has a viral infection and the antiviral immune response is enhanced.

[0129] The present invention also provides a method for inhibiting the growth of a tumor in a subject, the method comprising administering an anti-huTIGIT antibody of the present invention or an antigen-binding fragment thereof to the subject, such that the growth of the tumor is inhibited.

[0130] The present invention further provides a method of treating cancer, for example by immunotherapy, the method comprising administering a therapeutically effective amount of the anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention, for example as a pharmaceutical composition, to a subject in need thereof, thereby treating cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancer. In certain embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

[0131] In a seventh aspect, the present invention provides a method of treating a TIGIT-related disease, the method comprising administering an effective amount of the anti-TIGIT antibody or antigen-binding fragment thereof of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, the cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect to a subject in need thereof.

[0132] In an eighth aspect, the present invention provides a pharmaceutical composition for treating a TIGIT-related disease, wherein the pharmaceutical composition comprises the anti-TIGIT antibody or antigen-binding fragment thereof of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, or the cell of the fourth aspect. In combination with one or more additional therapeutic agents, or as a bispecific reagent together with one or more additional therapeutic agents, the therapeutic agents being, for example, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG3 antibody, anti-GITR antibody, anti-OX40 antibody, anti-CD73 antibody, anti-CD40 antibody, anti-CD137 mAb, anti-CD27 mAb, anti-CSF-1R antibody, and / or anti-CTLA-4 antibody, TLR agonists, or small molecule antagonists of IDO or TGFβ. In certain embodiments, anti-huTIGIT therapy is combined with anti-PD-1 and / or anti-PD-L1 therapy (for example, treatment with an antibody or antigen-binding fragment thereof that binds to human PD-1, or treatment with an antibody or antigen-binding fragment thereof that binds to human PD-L1).

[0133] The present invention also provides methods for detecting the presence of TIGIT in a sample, on cells within a sample (e.g., by FACS), or at specific locations in cells or tissues (e.g., by IHC), or for sorting cells based on the presence or absence of TIGIT on their surface (e.g., by FACS), the methods comprising contacting the sample with an anti-huTIGIT antibody or an antigen-binding fragment thereof of the present invention under conditions that permit the formation of a complex between the antibody or its antigen-binding fragment and TIGIT, and detecting the formation of the complex. In some embodiments, the anti-TIGIT antibody used for detection is conjugated to a detectable label.

[0134] The present invention uses a transgenic chromosome mouse (TC-mAb TM mouse) model in which a fully human antibody gene sequence (including gene regulatory sequences) has been transferred. The target antigen is used to immunize the transgenic chromosome mouse to directly obtain a fully humanized antibody. The obtained antibody does not require subsequent humanization and affinity modification, thereby reducing costs and shortening the development cycle. Additionally, the antibody is a fully human antibody derived from a transgenic chromosome mouse, which significantly reduces its immunogenicity and is more favorable for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 is a graph showing the results of huTIGIT binding affinity of the recombinant huTIGIT protein and the TC-mAb TM mouse-derived anti-TIGIT antibodies specified in the present invention. See Example 3.

[0136] Figure 2A and Figure 2B shows the results of epitope competition of the recombinant huTIGIT protein and the TC-mAb TM mouse-derived anti-TIGIT antibodies specified in the present invention. See Example 3.

[0137] Figure 3A is a graph showing the results of CHO-TIGIT binding assays at different concentrations of the TC-mAb TM mouse-derived anti-TIGIT antibodies and CHO cells expressing human TIGIT specified in the present invention.

[0138] Figure 3B is a graph showing the results of CHO-TIGIT binding assays at different concentrations of the TC-mAb TM mouse-derived anti-TIGIT antibodies and CHO cells expressing cynomolgus monkey TIGIT (mkTIGIT) specified in the present invention. See Example 4.

[0139] Figure 4It is a graph showing the results of a CHO-TIGIT CD155 blocking assay using CHO cells expressing human TIGIT and the TC-mAbTM mouse-derived anti-TIGIT antibody specified in the present invention at different concentrations. See Example 5.

[0140] Figure 5 A is a graph showing the inhibitory effect of the TC-mAb TM mouse-derived anti-TIGIT antibody 4A063 specified in the present invention in combination with an anti-PD1 antibody on tumor growth in a human TIGIT transgenic mouse model. Figure 5 B shows the average body weight of each group of mice as a function of time. See Example 6.

[0141] Figure 6 A is a graph showing the regulation of the proportion of tumor-infiltrating lymphocyte CD8+T (CD8+TIL) cells within the CD3+T cell population in CT26 tumors treated with the specified TC mAb TM derived anti-TIGIT antibody 4A063 in combination with an anti-PD1 antibody. See Example 7.

[0142] Figure 6 B is a graph showing the regulation of the proportion of CD8+T cells within the CD3+T cell population in splenocytes of human TIGIT transgenic mice bearing CT26 tumors treated with the specified TC mAb TM derived anti-TIGIT antibody 4A063 in combination with an anti-PD1 antibody. See Example 7. Detailed Description

[0143] Definition

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative and not intended to be limiting. All published publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0145] While numerical ranges and parameter approximations are disclosed herein in their broadest scope, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (including the end points); that is, all subranges beginning with a value of 1 or more (such as 1 to 6.1) and ending with a value of 10 or less (such as 5.5 to 10).

[0146] It should also be noted that, unless clearly and expressly limited to one referent, the singular forms as used in this specification include the plural of the referents. Unless the context indicates otherwise, the term “or” and the term “and / or” are used interchangeably.

[0147] As used herein, the term “comprising” or “including” means that the various components can be used together in the mixtures or compositions of the present invention. Thus, the terms “consisting essentially of” or “consisting of” are included within the term “comprising” or “including”.

[0148] As used herein, the terms "identity", "percent identity", "homology", or "identical" refer to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning the two sequences and refers to the number of identical residues (i.e., amino acids or nucleotides) at positions shared by the compared sequences. Standard algorithms in the art can be used (e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444) or computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI), which are publicly available in the form of BLAST and FASTA. Additionally, ENTREZ, available at the National Institutes of Health (Bethesda MD), can be used for sequence comparison. When using the BLAST or Gapped BLAST programs, the default parameters of each program can be used (e.g., BLASTN, available on the Internet site of the National Center for Biotechnology Information). In one embodiment, GCG with a gap weight of 1 can be used to determine the percent identity between two sequences. Each amino acid gap is given a weight as if it were a single amino acid mismatch between the two sequences. Alternatively, the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, CA) sequence alignment software package, can be used.

[0149] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0150] Conventional antibody structural units typically comprise tetramers. Each tetramer is typically composed of two pairs of identical polypeptide chains, each pair having one "light" chain and one "heavy" chain. Human light chains are classified as kappa light chains and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, and epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Thus, as used herein, "isotype" means any subclass of immunoglobulin defined by the chemical and antigenic characteristics of its constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. It should be understood that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses.

[0151] As used herein, the "CDR region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later). There are three heavy-chain CDRs and three light-chain CDRs. As used herein, the term CDR or CDRs is used to indicate one or several or even all of these regions, which contain most of the amino acid residues responsible for binding through the affinity between the antibody and the antigen or its epitope.

[0152] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of a full-length antibody and antibody mimetics that retains the ability to specifically bind to an antigen (e.g., TIGIT), and generally includes at least a portion of the antigen-binding region or variable region of the parent antibody. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., the variable domain of the heavy chain or the variable domain of the light chain). The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed in moles, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody for the target. Antibody fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, FD fragments, complementarity-determining region (CDR) fragments, disulfide-stabilized proteins (dsFv), etc.; linear antibodies, single-chain antibodies (e.g., scFv single antibodies) (technology from Genmab), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (e.g., VH domain antibodies), domain antibodies (technology from Ablynx); multispecific antibodies formed from antibody fragments (e.g., trispecific antibodies, tetravalent antibodies, etc.); and engineered modified antibodies, such as chimeric antibodies (humanized murine antibodies), heteroconjugate antibodies, etc. These antibody fragments are obtained by conventional techniques known to those skilled in the art, and the utility of these fragments is screened by the same methods as for full antibodies.

[0153] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of mammalian immunoglobulin heavy or light chains) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0154] In one embodiment, the antibodies of the present invention can be multispecific antibodies, particularly bispecific antibodies, which are sometimes also referred to as "diabodies". These are antibodies that bind to two (or more) different antigens or different epitopes on the same antigen. Diabodies can be made in a variety of ways known in the art, e.g., prepared chemically or from hybridomas.

[0155] As used herein, the term "transchromosomic mouse (TC-mAb TM mouse)" refers to a mouse containing a murine artificial chromosome that contains a human antibody heavy chain gene or locus and / or a human antibody κ light chain gene or locus and / or a human antibody λ light chain gene or locus, and at least two murine endogenous antibody genes or loci corresponding to the human antibody genes or loci are knocked out. TC-mAb TMMice and their offspring can stably maintain human antibody genes and produce human antibodies.

[0156] The antibodies of the present invention are generally isolated or recombinant. When "isolated" is used to describe the various polypeptides disclosed herein, it means a polypeptide that has been identified and separated and / or recovered from the cells or cell culture expressing the polypeptide. Generally, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities.

[0157] The present invention further provides variant antibodies. That is, many modifications can be made to the antibodies of the present invention, and these modifications include, but are not limited to, amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, other types of covalent modifications, etc. For example, "variant" herein means a polypeptide sequence that is different from the parental polypeptide sequence due to at least one amino acid modification. Amino acid modifications can include substitutions, insertions, and deletions. Generally, a variant can include any number of modifications as long as the function of the protein remains as described herein.

[0158] As used herein, the term "epitope" refers to a determinant that interacts with the specific antigen-binding site (termed the paratope) in the variable region of an antibody molecule. An epitope is a group of molecules (such as amino acids or sugar side chains) and usually has specific structural features as well as specific charge features. A single antigen can have more than one epitope.

[0159] An epitope can contain amino acid residues that are directly involved in binding (also termed the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide; in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide. An epitope typically includes at least 3, more usually at least 5, or 8 - 10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to the target antigen.

[0160] As used herein, the terms "polypeptide", "peptide", and "protein" are used interchangeably to refer to an amino acid polymer of any length of at least two amino acids.

[0161] As used herein, the terms "polynucleotide", "nucleic acid molecule", and "nucleic acid sequence" are used interchangeably herein to refer to a nucleotide polymer of any length of at least two nucleotides, and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and their variants.

[0162] As used herein, the terms "pharmaceutical composition", "combination drug", and "drug combination" are used interchangeably and mean a combination of at least one drug and an optionally pharmaceutically acceptable carrier or excipient for achieving a particular purpose. In certain embodiments, the pharmaceutical composition comprises combinations that are separated in time and / or space, provided that they are capable of working together to achieve the purposes of the present disclosure.

[0163] As used herein, "therapeutically effective amount" or "effective amount" means a dose sufficient to confer a benefit on the subject to which it is administered. The actual amount administered, as well as the rate and duration of administration, will depend on the condition and severity of the subject being treated. The treatment regimen (e.g., dose determination, etc.) is ultimately the responsibility and decision of the general practitioner and other physicians, who typically consider the disease to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.

[0164] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human, or non-human to whom treatment according to the methods of the present invention is provided. The present invention contemplates veterinary and non-veterinary applications. A human patient can be an adult or an adolescent (e.g., a person under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., domestic pigs, mini-pigs), horses, dogs, cats, cattle, and other domestic, farm, and zoo animals.

[0165] The antibodies and chemotherapeutic agents of the present invention are administered to a subject by known methods, such as intravenous administration in the form of a single dose or by continuous infusion over a period of time, intramuscular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes of administration.

[0166] As used herein, the term "pharmaceutically acceptable" means that the compound is physiologically acceptable when administered to a human and does not cause adverse reactions such as gastrointestinal disorders, dizziness, or other adverse reactions or systemic adverse reactions similar to these adverse reactions.

[0167] In the present disclosure, "pharmaceutically acceptable carriers" include, but are not limited to, binders (such as microcrystalline cellulose, alginate, gelatin, and polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose, and anhydrous lactose), disintegrants (such as cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose), lubricants (magnesium stearate, aluminum stearate, talc, polyethylene glycol, sodium benzoate), wetting agents (such as glycerol), surfactants (such as cetyl alcohol), as well as absorption enhancers, flavoring agents, sweetening agents, diluents, coating agents, and the like.

[0168] Unless otherwise indicated, the term TIGIT or "T cell immunoreceptor with Ig and ITIM domains" as used herein refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIGU, VSTM3, and WUCAM. The term encompasses "full-length", unprocessed TIGIT (e.g., full-length human TIGIT having the amino acid sequence of SEQ ID NO:), and any form of TIGIT. The term also encompasses naturally occurring TIGIT variants, such as splice variants or allelic variants. The term "TIGIT-related disease" refers to the abnormal expression of TIGIT protein or its ligand CD155 in a subject (e.g., human) in a tumor (e.g., melanoma, breast cancer, non-small cell lung cancer (NSCLC), colon adenocarcinoma (COAD), gastric cancer, acute myeloid leukemia (AML), and multiple myeloma (MM) (Clin Exp Immunol. 2020 May; 200(2): 108-119)) or in an immune-related disease (e.g., T cell dysfunctional disease). After an anti-TIGIT antibody blocks its binding to the ligand, the anti-TIGIT antibody can inhibit the growth of tumor cells, or alleviate the symptoms of other diseases, or cure the related diseases, thereby achieving a therapeutic effect on the diseases. Such diseases are defined as TIGIT-related diseases.

[0169] "T cell dysfunction disorder" is a T cell disorder or condition characterized by a reduced responsiveness to antigenic stimulation. In some embodiments, the T cell dysfunction disorder is characterized by T cell exhaustion. In certain embodiments, the T cell dysfunction disorder is a disorder significantly associated with an inappropriate reduction in signal transduction via OX40 and / or OX40L. In another embodiment, the T cell dysfunction disorder is a disorder in which T cells are unresponsive or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In certain aspects, the reduced responsiveness results in ineffective control of pathogens or tumors expressing immunogens. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity. In some embodiments, the subject is a human.

[0170] The terms "cancer" and "tumor" are used interchangeably. They refer to a large group of diseases characterized by uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or the invasion of cells into adjacent tissues, and may metastasize to distant parts of the body via the lymphatic system or blood flow. Cancers include benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers also include hematological malignancies.

[0171] "Hematological malignancies" include lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include B cell lymphomas and T cell lymphomas. B cell lymphomas include Hodgkin lymphoma and most non-Hodgkin lymphomas. Non-limiting examples of B cell lymphomas include diffuse follicular lymphoma, large B cell lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlapping with chronic lymphocytic leukemia), Burkitt lymphoma, mantle cell lymphoma (MCL), mediastinal large B cell lymphoma, Waldenström macroglobulinemia, nodular marginal zone B cell lymphoma, splenic marginal zone lymphoma, primary effusion lymphoma, intravascular large B cell lymphoma, lymphomatoid granulomatosis. Non-limiting examples of T cell lymphomas include extranodal T cell lymphoma, cutaneous T cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma. Hematological malignancies also include leukemias, such as but not limited to secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and acute lymphocytic leukemia. Hematological malignancies further include myelomas, such as but not limited to multiple myeloma and smoldering multiple myeloma. Other hematological and / or B cell or T cell-related cancers are encompassed by the term hematological malignancies.

[0172] The term "PVR-positive tumor" refers to a tumor in which the expression of PVR in cancer tissues is increased. PVR-positive tumors include, but are not limited to, adrenocortical carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, pheochromocytoma and paraganglioma, lung adenocarcinoma, head and neck squamous cell carcinoma, prostatic adenocarcinoma, endometrial carcinoma of the uterine corpus, cervical cancer, cutaneous melanoma, mesothelioma, urothelial bladder carcinoma, colorectal adenocarcinoma, clear cell renal cell carcinoma, lung squamous cell carcinoma, carcinosarcoma of the uterus, sarcoma, serous cystadenocarcinoma of the ovary, papillary thyroid carcinoma, glioblastoma multiforme, breast cancer, low-grade glioma, and diffuse B-cell lymphoma.

[0173] As used herein, the term "immune-related disease" refers to an immune-related disease in a mammal that is caused by, mediated by, or otherwise contributed to by components of the mammalian immune system, and also includes diseases that stimulate or interfere with the immune response and thereby may improve disease development. "Immune-related diseases" include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immune deficiency diseases, tumors, and the like.

[0174] The anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can be used to treat an infection or infectious disease in a subject (such as a human). In some preferred embodiment options, the infection or infectious disease is selected from viral infections, bacterial infections, fungal infections, and parasitic infections, including but not limited to HIV, hepatitis virus, herpes virus, CMV, EBV, and influenza.

[0175] Hereinafter, some preferred embodiments and aspects of the present invention will be further described in conjunction with specific examples, and these examples should not be construed as limiting the scope of the present invention.

[0176] Examples

[0177] Example 1 Generation of Anti-TIGIT Monoclonal Antibody

[0178] 1. TIGIT Recombinant Protein for Antigen Immunization and Binding Assay

[0179] Synthesize cDNA encoding full-length human TIGIT (huTIGIT, SEQ ID NO: 1) based on the GenBank sequence (locus: NM_173799), and the cDNA was purchased from Eurofins. Amplify the coding region of the extracellular domain ECD corresponding to amino acids (AA) 1 - 141 (SEQ ID NO: 2) of full-length human TIGIT by PCR and clone it into an expression vector to generate two recombinant fusion protein Trx-huTIGIT-HIS and Gst-huTIGIT-HIS expression plasmids, respectively. To produce the recombinant fusion proteins, transfer the recombinant fusion protein (Trx-TIGIT-HIS and Gst-TIGIT-HIS) expression plasmids into competent Escherichia coli (E. coli gamiB(DE3)pLysS | Novagen) and culture them. After IPTG induction, centrifuge the E. coli cells to collect the pellet. After sonicating the E. coli, obtain the pellet by centrifugation. Add solubilization reagents to dissolve the pellet, then purify it through a Ni-NTA column (Qiagen, Ni-NTA Superflow, #30410) and dialyze it. Detect the purification effect of the recombinant protein by PAGE, and then store the recombinant protein in small aliquots at -30 °C.

[0180] ATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAATCAGCTGGACAGGAGGAATGGAGCCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAGAGACTGGTTAG(SEQ ID NO:1);

[0181] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAI CNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTY TGRIFLEVLESSVAEHGAR FQIP(SEQ ID NO:2).

[0182] 2. Establishment of a stable expression cell line

[0183] The full-length cDNAs encoding human TIGIT (huTIGIT, SEQ ID NO:1) and cynomolgus macaque TIGIT (mkTIGIT, SEQ ID NO:3) were synthesized separately based on the GenBank sequences (NM_73799) and (XM_005548101.2), and the cDNAs were purchased from Genscript. After PCR amplification, the DNA products were cloned into the pcDNA3.1 expression vector (Invitrogen) and transduced into the CHO-K1 cell line (JCRB, #JCRB9018) to generate the CHO-huTIGIT and CHO-mkTIGIT cell lines. The HuTIGIT or mkTIGIT highly-expressing stable cell lines were selected by culturing in medium with G418, eGFP expression, and FACS binding assay.

[0184] ATGCGGTGGTGTCTCTTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAAAGAAAGGTGGCTCTGTTATCTTACAATGTCACCTCTCCTCCACCATGGCACAAGTGACCCAGGTCAACTGGGAGCAGCATGACCATTCGCTTCTGGCCATTCGTAATGCTGAGTTGGGGTGGCACATCTACCCAGCCTTCAAGGATCGAGTGGCCCCGGGTCCTGGCCTGGGCCTCACCCTCCAGTCGCTGACCATGAATGATACAGGGGAGTACTTCTGCACCTATCACACCTACCCTGATGGGACTTACAGAGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACAGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCATGATGCTGGTGGTCATCTGCATAGCAGTCATCGTGGTGGTCGTGTTGGCTAGAAAGAAGAAATCCCTCAGAATCCATTCTGTGGAAAGTGGCCTCCAGAGAAAATCAACTGGACAGGAAGAACAGATTCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCAGGGAGATGACTGTG CCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGGAGCTGCAGCTTCTTCACAGAGACTGGGTAG(SEQ ID NO:3).

[0185] 3. Immunization, Hybridoma Fusion and Cloning

[0186] The human TIGIT recombinant protein (Trx-huTIGIT-HIS fusion protein, 100 μg / mouse / primary, 50 μg / mouse / boost) was mixed with Freund's complete adjuvant (FCA, purchased from BD, catalog number 263810, 100 μL / mouse / primary) for the primary immunization injection of transchromosomal mice (TC-mAb TM mice) at 6 - 8 weeks of age, and (Sigma Adjust SAS, purchased from sigma, catalog number s6322-1vl, 50 μL / mouse / boost) was used for boost immunization injection, with an interval of 2-3 weeks. The last immunization did not require adjuvant, only the Trx-huTIGIT-HIS fusion protein (50 μg / mouse / final) was needed. All immunizations were performed by intraperitoneal injection.

[0187] Three days after the last immunization, the mice were euthanized, the spleen and lymph nodes were aseptically removed, and mouse lymphocytes were aseptically isolated and extracted. The obtained lymphocyte population was fused with mouse myeloma cells (1:1, P3X63-Ag8.653, ATCC, #CRL-1580) by electrofusion. The fused cells were placed in HAT medium in a 96-well plate and incubated at 37 °C and 5% CO2 for 7 days, then changed to HT medium and incubated for 5 days.

[0188] 4. Evaluate the binding activity of anti-TIGIT antibodies by ELISA, immunocytochemistry (ICC) and flow cytometry

[0189] The user uses the TIGIT protein to screen the supernatant containing specific anti-TIGIT antibodies by ELISA. A 96-well plate (Nunc, catalog number 44-2404) is coated with Gst-huTIGIT-HIS fusion protein and Trx-huTIGIT-HIS (50 ng / well) respectively, diluted with PBS buffer, and incubated overnight at 4°C. Then, the wells are sealed with 300 μL of TBS containing skim milk and Tween-20 for 0.5 hours at room temperature. After washing, 100 μL of supernatant and / or serum is added and incubated at room temperature. To detect the specificity of the antibody, a horseradish peroxidase-conjugated anti-human IgG antibody (goat anti-human IgG-Fc fragment cross-adsorbed antibody HRP-conjugated, BETHYL, #A80-304P) is diluted to the optimal concentration in PBS containing 0.05% Tween-20, then added at 100 μL / well after washing and incubated for 0.5 hours at room temperature. The plate is washed 3 times with 300 μL of TBS containing 0.05% Tween-20. 100 μL of substrate solution containing 0.5 mg / mL OPD and 0.03% H2O2 is added, the plate is incubated for 30 minutes at room temperature, then 25 μL of 1M H2SO4 (Nacalai Tesque, #95626-06) is added, and then read at 492 nm. Positive well clones are selected and inoculated into a new 96-well plate. After 3 days, the supernatant of the new 96-well plate is screened by ELISA using the human TIGIT protein. The hybridoma cell lines binding to human TIGIT are expanded and cultured for 2-4 days, and the ELISA-positive clones are detected by ICC and flow cytometry using CHO-huTIGIT and CHO-mkTIGIT cells, and the positive clones are selected. After culturing for several days, a secondary detection is performed according to the above method. The secondary positive clones are diluted to the limit, and ICC and flow cytometry are tested again two weeks later, and limiting dilution is performed again.

[0190] Example 2 Sequence Analysis of Anti-TIGIT Antibodies

[0191] After preliminary screening by ELISA, ICC, and FACS, the positive hybridoma clones are subcloned by limiting dilution. After re-verification, the cloned hybridoma cells are cultured in a 10 cm culture dish. When the cell density reaches 80% to 90%, the cells are collected and suspended in a solution. Using a microKit (QIAGEN |#74104) RNA was extracted from suspension cells. The extracted RNA was rapidly amplified at the 5' cDNA end using a kit (TaKaRa, #Z4858N). Sequence analysis of the product (Eurofins) is shown in Table 1. Based on the sequence, a TIGIT antibody expression plasmid was constructed and expressed in HEK293 cells. Eight antibodies were purified by Protein A and analyzed by SDS-PAGE, and the purity of the antibodies exceeded 95%. The ten obtained antibodies were subjected to amino acid sequencing, and the heavy chain variable region (VH) and light chain variable region (VL) sequences are shown in Table 1.

[0192] Table 1 TC mAb in the present invention TM Variable region sequences of the VH and VL regions of mouse-derived anti-TIGIT antibodies

[0193]

[0194]

[0195]

[0196]

[0197] CDR prediction of VL and VH was performed using the IMGT program, and the results are shown in Table 2.

[0198] Table 2 Predicted CDRs of eight antibodies

[0199]

[0200]

[0201] It should be noted that when using different CDR prediction programs, the CDRs of the same VH or the same VL may show slight differences, such as changes in amino acid positions. These different CDRs based on the same VH or VL are also within the scope of the present invention.

[0202] TC-mAb specified in Example 3 TM Determination of the affinity between mouse-derived anti-TIGIT antibody and recombinant human TIGIT protein

[0203] The equilibrium dissociation constants (KD) of the eight exemplary antibodies of the present invention that bind to human TIGIT were measured by Fortebio Octet RED96. The measurement method was carried out according to the existing method (Estep, P et al., High-throughput solution Based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2):p.270-8). For the TC-mAb specified in the present invention TM The affinity between the mouse-derived anti-TIGIT antibody and TIGIT-HIS (Biointron, BI120) was measured. An NTA (HIS-tag) sensor was applied. After the sensor was equilibrated in the assay buffer, human TIGIT-HIS was loaded onto the NTA sensor (fortebio) for affinity measurement. The sensor loaded with the antigen was placed in a solution containing the antibody (antibody concentrations were 5, 2.5, 0.83, 0.278, 0.09, 0.03, and 0.01 μg / ml respectively) until a plateau was reached, and then the sensor was transferred to the assay buffer for dissociation for at least 2 minutes for dissociation rate measurement. A 1:1 binding model was used for kinetic analysis.

[0204] In the experiment described above, the TC-mAb specified in the present invention TM The KD values of the mouse-derived anti-TIGIT antibodies are shown in Table 3.

[0205] Table 3 Monovalent KD of eight antibodies

[0206]

[0207] Compared with the anti-TIGIT reference antibody Tiragolumab (synthesized according to the Tiragolumab sequence disclosed in the KEGG-DRUG database), the Octet binding assay was used to study the epitopes of all human TIGIT antibodies (1B2-8C, 4A042-H3, 4A042-H7, 4B030a, 4B037a, 4B056a, 4A063, 4D035a, 4E061a) that bind to human TIGIT. The experimental procedure was as follows: The sensor loaded with TIGIT-HIS (Biointron, BI120) was placed in a solution containing the TIGIT antibody until a plateau was reached, then the sensor was transferred to the assay buffer until saturation, and then the sensor was transferred to other analytes or buffers containing the reference antibody (Tiragolumab). After binding reached a plateau, it was eluted. Epitope grouping showed that there was competition among the three candidate clone epitopes compared with Tiragolumab, that is, they combined with the same antigenic epitope of TIGIT. The results are shown in Figure 2A in.

[0208] There is epitope competition among multiple antibodies in the present invention. 1B2-8C can block the binding of itself and Tiragolumab, 4A042-H3, 4A042-H7, and 4B030a, partially block the binding of 4A063, but do not block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; Tiragolumab and 4A042-H3 can block the binding of Tiragolumab, 1B2-8C, 4A042-H7, and 4B030a, and do not block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; 4A042-H7 can block the binding of itself and Tiragolumab, 1B2-8C, 4A042-H3, and 4B030a, partially block the binding of 4B037a and 4A063, but do not block the binding of 4B056a, 4D035a, and 4E061a; 4B030a can block its own binding, partially block the binding of 4B056a and 4A063, and do not block the binding of 4B037a, 4D035a, and 4E061a; 4B037a and 4A063 can block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a, and partially block the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a; 4B056a can block the binding of itself and 4B037a, 4A063, 4D035a, and 4E061a, partially block the binding of Tiragolumab, 1B2-8C, 4A042-H3, and 4B030a, but do not block the binding of 4A042-H7; 4D035a and 4E061a can block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a, and do not block the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a. The results are shown in Figure 2B and 2C in.

[0209] TC-mAb specified in Example 4 TM Binding activity of mouse-derived anti-TIGIT monoclonal antibody to cell surface TIGIT

[0210] CHO-huTIGIT cells or CHO-mkTIGIT cells were seeded in 96-well plates. The antibodies of Example 2 were diluted to different concentrations and added to the 96-well plates covered with cells (100 μL / well), and incubated at 4 °C (on ice) for 1 hour.

[0211] Wash the cells by adding 200 μL / well of wash buffer, centrifuge at 1600 rpm (about 260×g) for 3 minutes at 4 °C, and discard the supernatant. Repeat twice. Add 30 μL of wash buffer containing anti-human IgG secondary antibody (Jackson ImmunoResearch, #109-585-190, Alexa 594 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific) to each well, and incubate the cells at 4 °C (on ice) for 1 hour. Wash twice, transfer the cells to a flat-bottom 96-well plate, and analyze the cells with CytoFLEX S. Plot the antibody concentration with the logarithm of 10 as the abscissa and the median fluorescence value of the two channels as the ordinate. Compare the EC 50 (CHO-huTIGIT and CHO-mkTIGIT) with the peak of the curve. (μg / mL) The results are shown in Table 4 and Figures 3A-3B .

[0212] Table 4 Binding activity between the antibody and human TIGIT or cynomolgus monkey TIGIT (EC 50 , μg / mL)

[0213]

[0214]

[0215] Example 5 TC-mAb in the present invention TM Mouse-derived anti-TIGIT monoclonal antibody blocks the binding of TIGIT and CD155

[0216] Seed CHO-huTIGIT-eGFP cells in a V-bottom 96-well plate. Dilute the antibody of Example 2 and the reference antibody to different concentrations respectively, and add (100 μL / well) to the 96-well plate covered with cells (100%), and incubate at 4 °C for 1 hour.

[0217] Wash the cells by adding 200 μL / well of wash buffer and centrifuge at 1600 rpm (approx. 260×g) for 3 minutes at 4°C, and discard the supernatant. Repeat twice. Add 30 μL of wash buffer containing biotinylated human CD155 (human CD155 / PVR / NECL5 protein (Fc tag), biotinylated, Sin Biological, 10109-H02H-B), then incubate the cells at 4°C (on ice) for 1 hour. Add 30 μL of wash buffer containing streptavidin-594 (f.c. = 10 μg / ml) to the wells, then incubate the cells at 4°C for 30 minutes. Wash once, and transfer the cells to a flat-bottom 96-well plate, and analyze the cells using CytoFLEX S. Use the logarithm to the base 10 of the antibody concentration (μg / mL) as the abscissa, and use the median fluorescence value corresponding to each antibody concentration as the ordinate. By analyzing the IC 50 to distinguish the blocking ability of different antibodies to block the binding between CD155 and TIGIT.

[0218] Use IgG1 as a negative control antibody to test the blocking ability of eight antibodies against CD155. IC 50 (μg / mL) results are shown in Table 5 and Figure 4 in.

[0219] Table 5 Specified TC-mAb TM Blocking ability of mouse-derived anti-TIGIT monoclonal antibodies to block the binding of CD155 to TIGIT (IC 50 )

[0220]

[0221]

[0222] Example 6 In Vivo Efficacy of Combination Therapy of Anti-TIGIT Antibody and Anti-PD-1 Antibody

[0223] To evaluate the synergistic effect of anti-TIGIT antibody and anti-PD-1 antibody in vivo, the mouse colorectal cancer cell line CT26.WT (5×10 5 cells / mouse) was subcutaneously transplanted into TIGIT humanized BALB / c mice. When the average tumor volume reached 120 ± 50 mm 3At that time, mice were randomly grouped according to tumor volume (n = 3), and an IgG negative control antibody (anti-HEL human IgG1 isotype, biointron, 200 μg / mouse / time), an anti-mPD-1 antibody (InVivoMAb anti-mouse PD-1, lot number: 795720D1, 20 μg / mouse / time), an anti-mPD-1 antibody (20 μg / mouse / time) combined with a positive reference antibody (Tiragolumab-hIgG1, 200 μg / mouse / time), and an anti-mPD-1 antibody (20 μg / mouse / time) combined with a 4A063 antibody (4A063-hIgG1, 200 μg / mouse / time) were administered once every three days for a total of 6 times. Tumor volume and body weight were measured twice a week.

[0224] The results show that in Figure 5 Among them, Figure 5 the average tumor volume of each group of mice as a function of time and the average weight of each group of mice as a function of time were provided. Compared with the positive reference antibody, 4A063 showed significant inhibition of tumor growth (TGI: 44% vs. 92.33%), and there was no significant difference in the average body weight among the groups.

[0225] Example 7 Characterization of the mechanism of action of the combination therapy of anti-TIGIT antibody and anti-PD-1 antibody in vivo anti-tumor activity

[0226] To study the in vivo mode of action of the anti-TIGIT antibody, after combination treatment with the anti-TIGIT antibody 4A063 (hIgG1) and the anti-PD-1 antibody, the immune cell infiltration of the tumor was analyzed by flow cytometry. As described in Example 6, mice were inoculated and treated. Three days after six treatments, the mice were sacrificed and tumors and spleens were collected. Tumors were dissociated with tumor digestion buffer (1 mg / mL collagenase I and 20 μg / mL DNAase I, sigma), and single cell suspensions of spleens were obtained directly after grinding the spleens. After staining with Fc-block, the cells were stained with anti-CD3 (FITC anti-mouse CD3, Biolegend, 100204), anti-CD8 (PE anti-mouse CD8a, Biolegend, 100708). After fixation and permeabilization with a commercial buffer (BD Cytofix / Cytoperm TM Fixation / Permeabilization Kit, 554714), the cells were stained with anti-IFNγ antibody (PerCP / Cyanine5.5 anti-mouse IFN-γ, Biolegend, 505822). After conventional washing and filtration, the cells were analyzed by flow cytometry.

[0227] The results show that in Figure 6 Among them.Figure 6 Panel A shows that in vivo treatment of tumors with the anti-TIGIT antibody 4A063hIgG1 led to an increased proportion of the CD8+ TIL population in the tumor microenvironment of the combination therapy compared to the control group (P = 0.0335), which was not observed in the single PD-1 antibody group. At the same time, the flow cytometry results of T cells in splenocytes (shown in Figure 6 Panel B) were consistent with the results in the tumor microenvironment, which also showed that the proportion of the CD8+ TIL population in the combination therapy group was significantly higher than that in the isotype control group (P = 0.0025), and also higher than that in the single PD-1 antibody treatment group (P = 0.0171). This indicates that cytotoxic effector T cells were activated after combination therapy, which may explain the difference between the combination therapy group and the single PD-1 group, as discussed in Example 5. In the comparison within the combination therapy group, the increase in CD8 + TIL was more significant in the 4A063-hIgG1 combination group compared to the combination group with the anti-TIGIT reference antibody (Tiragolumab-hIgG1). For the PD-1 antibody combined with the anti-TIGIT antibody 4A063 hIgG1 treatment group, the function of intratumoral T cells was also improved, and the production of IFNγ by CD3+CD8+ T cells increased.

[0228] All publications and patents cited in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions described in this invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, many variations of the described modes for carrying out the invention are intended to be included within the scope of the claims, which will be apparent to those skilled in the relevant art.

Claims

1. An anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, which comprises a heavy chain variable region and a light chain variable region, wherein, (I) The heavy chain variable region has CDRH1, CDRH2, and CDRH3 with amino acid sequences as shown in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 respectively, and the light chain variable region has CDRL1, CDRL2, and CDRL3 with amino acid sequences as shown in SEQ ID NO:44, LGS, and SEQ ID NO:46 respectively; or, (II) The heavy chain variable region has CDRH1, CDRH2, and CDRH3 with amino acid sequences as shown in SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67 respectively, and the light chain variable region has CDRL1, CDRL2, and CDRL3 with amino acid sequences as shown in SEQ ID NO:62, LGS, and SEQ ID NO:64 respectively.

2. The anti-TIGIT antibody or an antigen-binding fragment thereof according to claim 1, which comprises a heavy chain variable region and a light chain variable region, wherein, (I) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:12; or, (II) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

18.

3. The anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 or 2, wherein, The anti-TIGIT antibody or its antigen-binding fragment further comprises a heavy chain constant region, and the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

4. The anti-TIGIT antibody or an antigen-binding fragment thereof according to claim 3, wherein, The heavy chain constant region is the heavy chain constant region of human IgG.

5. The anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 or 2, wherein, The anti-TIGIT antibody or its antigen-binding fragment is in a form selected from the group consisting of: F(ab')2, Fab', Fab, Fv, scFv, and combinations thereof.

6. The anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 or 2, wherein, The anti-TIGIT antibody or its antigen-binding fragment is conjugated to a reagent, wherein the reagent is a detectable label or a cytotoxic agent.

7. A polynucleotide, wherein, The polynucleotide encodes the anti-TIGIT antibody or its antigen-binding fragment according to any one of claims 1 to 6.

8. An expression vector, wherein, The expression vector enables the expression of the anti-TIGIT antibody or its antigen-binding fragment according to any one of claims 1 to 6.

9. An engineered cell, wherein, The engineered cell comprises the polynucleotide according to claim 7 or the expression vector according to claim 8.

10. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the anti-TIGIT antibody or its antigen-binding fragment according to any one of claims 1 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8, or the engineered cell according to claim 9, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, wherein, The pharmaceutical composition further comprises another immune checkpoint inhibitor.

12. The pharmaceutical composition according to claim 11, wherein, The another immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor and / or a CTLA-4 inhibitor.

13. The pharmaceutical composition according to claim 11, wherein, The another immune checkpoint inhibitor is an antibody or its antigen-binding fragment directed against another immune checkpoint.

14. Use of the anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8, the engineered cell according to claim 9, or the pharmaceutical composition according to any one of claims 10 - 13 for the manufacture of a medicament for the treatment of TIGIT-related diseases, wherein, The TIGIT-related disease is a tumor selected from the group consisting of melanoma, breast cancer, non-small cell lung cancer, colorectal adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma.

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