Tigit antibodies and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of Chinese Invention Patent Application No. 2022800819862, entitled "TIGIT Antibodies and Uses Thereof."

[0002] Cross Reference to Related Applications

[0003] This application claims priority to PCT Application No. PCT / CN2021 / 139122, filed December 17, 2021, entitled "TIGIT Antibodies and Uses Thereof," the disclosure of which is incorporated herein by reference. TECHNICAL FIELD

[0004] The present application relates to antibodies or antigen-binding fragments thereof that specifically bind to TIGIT (T cell immunoreceptor with Ig and ITIM domains), pharmaceutical compositions comprising anti-TIGIT antibodies or antigen-binding fragments thereof, and uses thereof. BACKGROUND

[0005] In recent years, tumor immunotherapy has made great breakthroughs and become a new hope for tumor therapy. In particular, treatment programs that block tumor immune suppression checkpoints represented by PD-1 / PD-L1 and CTLA-4 have attracted much attention. Since 2000, the FDA has successively approved the use of PD-1 / PD-L1 monoclonal antibodies for the clinical treatment of malignant tumors such as melanoma, non-small cell lung cancer, and prostate cancer, and has achieved good therapeutic effects. However, the clinical response rate of PD-1 / PD-L1 monoclonal antibody therapy is still limited, which greatly limits its clinical application. Therefore, finding new immune suppression checkpoints has become a research hotspot.

[0006] TIGIT is a new immune suppression 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, including 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 TIGIT ligands found include CD155, CD112 and CD113, of which the main ligand of TIGIT is CD155. Crystal structure analysis shows that TIGIT and CD155 form homodimers, respectively, which further form heterotetramers 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 and macrophages, and non-hematopoietic tissues such as kidney, nervous system and small intestine. Similar to TIGIT, the activated receptors DNAM-1 and CD96 can also bind to CD155, but their affinity is weaker than that of TIGIT. In summary, the ligand-receptor mode of TIGIT / CD155 is similar to that of CTLA-4 / CD28 pathway. The inhibitory receptor with high affinity and the activated receptor with low affinity compete to bind to the same ligand, thereby precisely regulating the immune response. TIGIT binding to CD155 can exert an immunosuppressive effect by regulating DC function, inhibiting effector T cell activity, interfering with DNAM-1 co-activation and increasing Treg suppression (Clinical and Experimental Immunology, 2020 May; 200(2): 108-119, Immunity 40, 569-581).

[0007] Several human and mouse-based studies show that TIGIT is highly expressed in tumor-infiltrating lymphocytes. TIGIT is upregulated in many malignancies, including melanoma, breast cancer, non-small cell lung cancer, colon 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 +Expression of TIGIT in T cells and NK cells is generally consistent with high expression of other inhibitory receptors such as PD-1, LAG-3, Tim-3, and low expression of DNAM-1. High expression of TIGIT is often associated with poor prognosis of malignant tumors. High expression of TIGIT in NK cells is associated with the severity of the disease. Tumor growth is significantly reduced in TIGIT knockout mice, and survival rate is increased.

[0008] Due to its macromolecular properties, antibody drugs are often accompanied by immune-related adverse events (irAEs). In TIGIT knockout mice, there are no spontaneous autoimmune symptoms, and there are no hematopoietic cell development disorders. Only after crossing with mice with a tendency for autoimmune diseases, the incidence of autoimmune diseases increases. Compared with PD-1 and CTLA-4 mAbs, animal experiments show that the incidence of irAEs is lower during anti-TIGIT mAb administration (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 anticancer drugs.

[0009] Published clinical trial results show that the combination of TIGIT antibodies and PD-1 / PD-L1 monoclonal antibodies can significantly improve the response rate of patients, improve treatment effects, 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 worldwide, so it is necessary to develop antibodies with high affinity and activity as candidate drugs. SUMMARY

[0010] After a large number of experiments, the inventors of the present application used a transchromosomal mouse (TC mAb TM mouse) platform with all human antibody gene sequences to screen and unexpectedly obtain antibodies that specifically bind to TIGIT, which show excellent affinity for TIGIT and have potential prospects for drug development.

[0011] The present invention provides improved drugs and methods of treatment for cancer and chronic viral infections comprising anti-TIGIT antibodies or antigen-binding fragments thereof that specifically bind to human TIGIT (huTIGIT). Provided herein are isolated antibodies, such as monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to huTIGIT and have desirable functional attributes, such as high affinity specific binding to huTIGIT, binding to monkey TIGIT (e.g., cynomolgus monkey TIGIT), the ability to block TIGIT binding to PVR and Nectin-2, the ability to block TIGIT interaction with DNAM, or any combination of these attributes.

[0012] 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 binding of antibodies having the heavy and light chain variable domain sequences disclosed herein from binding to huTIGIT.

[0013] 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 costimulation of NK cells to increase NK-mediated anti-tumor responses killing. In another embodiment, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention deplete a population of regulatory T cells within a tumor that would otherwise suppress an anti-tumor immune response. In another embodiment, the anti-TIGIT antibodies of the present invention in the form of IgGl deplete CD8+ exhausted T cells and Tregs, allowing influx of fresh non-exhausted CD8+ T cells. In some embodiments, the anti-TIGIT antibodies of the present invention in the form of IgGl increase the proportion of 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.

[0014] In certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention do not bind to activating Fc gamma receptors (FcγRs), e.g., in embodiments that rely on enhancing anti-tumor activity of TIGIT-expressing cells. In alternative embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention bind to one or more activating FcγRs, e.g., in embodiments that rely on killing of TIGIT-expressing cells, such as exhausted CD8+ T cells or Tregs.

[0015] In a first aspect, the present application 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 a CDRH1, a CDRH2, and a CDRH3, and the light chain variable region comprises a CDRL1, a CDRL2, and a CDRL3.

[0016] In some embodiments of the present application, wherein

[0017] (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;

[0018] (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

[0019] (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.

[0020] In some other embodiments of the application, wherein

[0021] (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;

[0022] (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

[0023] (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.

[0024] In some other embodiments of the application, wherein

[0025] (a) the CDRH1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical 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;

[0026] (b) the CDRH2 comprises a sequence with 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

[0027] (c) the CDRH3 comprises a sequence with 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

[0028] (d) the CDRL1 comprises a sequence with 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;

[0029] (e) the CDRL2 comprises a sequence with 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

[0030] (f) the CDRL3 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical 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.

[0031] In some other embodiments of the application, wherein

[0032] (a) 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;

[0033] (b) 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;

[0034] (c) 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;

[0035] (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;

[0036] (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

[0037] (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.

[0038] In some other embodiments of the application, wherein the heavy chain variable region comprises a CDRH1, a CDRH2, and a CDRH3, and wherein

[0039] (a) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 23, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 24, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 25;

[0040] (b) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 29, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 30, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 31;

[0041] (c) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 35, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 36, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 37;

[0042] (d) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 41, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 42, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 43;

[0043] (e) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 47, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 48, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 49;

[0044] (f) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 53, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 54, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 55;

[0045] (g) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 59, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 60, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 61 ;

[0046] (h) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 65, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 66, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 67;

[0047] (i) the CDRH1 comprises a sequence of SEQ ID NO: 75, the CDRH2 comprises a sequence of SEQ ID NO: 76, and the CDRH3 comprises a sequence of SEQ ID NO: 77; or

[0048] (j) the CDRH1 comprises a sequence of SEQ ID NO: 81, the CDRH2 comprises a sequence of SEQ ID NO: 82, and the CDRH3 comprises a sequence of SEQ ID NO: 83.

[0049] In some other embodiments of the application, wherein the light chain variable region comprises a CDRL1, a CDRL2, and a CDRL3, and wherein

[0050] (a) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 20, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 21, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 22;

[0051] (b) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 26, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 27, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 28;

[0052] (c) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 32, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 33, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 34;

[0053] (d) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 38, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 39, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 40;

[0054] (e) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 44, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 45, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 46;

[0055] (f) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 50, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 51, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 52;

[0056] (g) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 56, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 57, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 58;

[0057] (h) the CDRL1 comprises a sequence as set forth in SEQ ID NO: 62, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 63, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 64;

[0058] (i) the CDRL1 comprises a sequence of SEQ ID NO: 72, the CDRL2 comprises a sequence of SEQ ID NO: 73, and the CDRL3 comprises a sequence of SEQ ID NO: 74; or

[0059] (j) the CDRL1 comprises a sequence of SEQ ID NO: 78, the CDRL2 comprises a sequence of SEQ ID NO: 79, and the CDRL3 comprises a sequence of SEQ ID NO: 80.

[0060] In some other embodiments of the application, wherein

[0061] (a) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 23, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 24, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 25; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 20, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 21, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 22;

[0062] (b) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 29, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 30, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 31; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 26, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 27, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 28;

[0063] (c) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 35, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 36, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 37; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 32, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 33, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 34;

[0064] (d) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 41, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 42, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 43; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 38, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 39, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 40;

[0065] (e) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 47, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 48, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 49; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 44, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 45, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 46;

[0066] (f) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 53, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 54, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 55; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 50, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 51, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 52;

[0067] (g) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 59, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 60, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 61; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 56, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 57, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 58;

[0068] (h) the CDRH1 comprises a sequence as set forth in SEQ ID NO: 65, the CDRH2 comprises a sequence as set forth in SEQ ID NO: 66, and the CDRH3 comprises a sequence as set forth in SEQ ID NO: 67; and the CDRL1 comprises a sequence as set forth in SEQ ID NO: 62, the CDRL2 comprises a sequence as set forth in SEQ ID NO: 63, and the CDRL3 comprises a sequence as set forth in SEQ ID NO: 64;

[0069] (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

[0070] (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.

[0071] In some other embodiments of the application, wherein

[0072] (a) the sequence of CDRH1 is set forth in SEQ ID NO: 23, the sequence of CDRH2 is set forth in SEQ ID NO: 24, and the sequence of CDRH3 is set forth in SEQ ID NO: 25; and the sequence of CDRL1 is set forth in SEQ ID NO: 20, the sequence of CDRL2 is set forth in SEQ ID NO: 21, and the sequence of CDRL3 is set forth in SEQ ID NO: 22;

[0073] (b) the sequence of CDRH1 is set forth in SEQ ID NO: 29, the sequence of CDRH2 is set forth in SEQ ID NO: 30, and the sequence of CDRH3 is set forth in SEQ ID NO: 31; and the sequence of CDRL1 is set forth in SEQ ID NO: 26, the sequence of CDRL2 is set forth in SEQ ID NO: 27, and the sequence of CDRL3 is set forth in SEQ ID NO: 28;

[0074] (c) the sequence of CDRH1 is set forth in SEQ ID NO: 35, the sequence of CDRH2 is set forth in SEQ ID NO: 36, and the sequence of CDRH3 is set forth in SEQ ID NO: 37; and the sequence of CDRL1 is set forth in SEQ ID NO: 32, the sequence of CDRL2 is set forth in SEQ ID NO: 33, and the sequence of CDRL3 is set forth in SEQ ID NO: 34;

[0075] (d) the sequence of CDRH1 is set forth in SEQ ID NO:41, the sequence of CDRH2 is set forth in SEQ ID NO:42, and the sequence of CDRH3 is set forth in SEQ ID NO:43; and the sequence of CDRL1 is set forth in SEQ ID NO:38, the sequence of CDRL2 is set forth in SEQ ID NO:39, and the sequence of CDRL3 is set forth in SEQ ID NO:40;

[0076] (e) the sequence of CDRH1 is set forth in SEQ ID NO:47, the sequence of CDRH2 is set forth in SEQ ID NO:48, and the sequence of CDRH3 is set forth in SEQ ID NO:49; and the sequence of CDRL1 is set forth in SEQ ID NO:44, the sequence of CDRL2 is set forth in SEQ ID NO:45, and the sequence of CDRL3 is set forth in SEQ ID NO:46;

[0077] (f) the sequence of CDRH1 is set forth in SEQ ID NO:53, the sequence of CDRH2 is set forth in SEQ ID NO:54, and the sequence of CDRH3 is set forth in SEQ ID NO:55; and the sequence of CDRL1 is set forth in SEQ ID NO:50, the sequence of CDRL2 is set forth in SEQ ID NO:51, and the sequence of CDRL3 is set forth in SEQ ID NO:52;

[0078] (g) the sequence of CDRH1 is set forth in SEQ ID NO:59, the sequence of CDRH2 is set forth in SEQ ID NO:60, and the sequence of CDRH3 is set forth in SEQ ID NO:61; and the sequence of CDRL1 is set forth in SEQ ID NO:56, the sequence of CDRL2 is set forth in SEQ ID NO:57, and the sequence of CDRL3 is set forth in SEQ ID NO:58;

[0079] (h) the sequence of CDRH1 is set forth in SEQ ID NO:65, the sequence of CDRH2 is set forth in SEQ ID NO:66, and the sequence of CDRH3 is set forth in SEQ ID NO:67; and the sequence of CDRL1 is set forth in SEQ ID NO:62, the sequence of CDRL2 is set forth in SEQ ID NO:63, and the sequence of CDRL3 is set forth in SEQ ID NO:64;

[0080] (i) the sequence of CDRH1 is set forth in SEQ ID NO:75, the sequence of CDRH2 is set forth in SEQ ID NO: 76, and the sequence of CDRH3 is set forth in SEQ ID NO: 77; and the sequence of CDRL1 is set forth in SEQ ID NO: 72, the sequence of CDRL2 is set forth in SEQ ID NO: 73, and the sequence of CDRL3 is set forth in SEQ ID NO: 74; or

[0081] (j) the sequence of CDRH1 is set forth in SEQ ID NO: 81, the sequence of CDRH2 is set forth in SEQ ID NO: 82, and the sequence of CDRH3 is set forth in SEQ ID NO: 83; and the sequence of CDRL1 is set forth in SEQ ID NO: 78, the sequence of CDRL2 is set forth in SEQ ID NO: 79, and the sequence of CDRL3 is set forth in SEQ ID NO: 80.

[0082] In some other embodiments of the application, 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.

[0083] In some other embodiments of the application, 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.

[0084] In some other embodiments of the application, wherein

[0085] (a) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 5, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 4;

[0086] (b) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 6;

[0087] (c) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 9, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 8;

[0088] (d) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 11, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 10;

[0089] (e) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 13, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 12;

[0090] (f) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 14;

[0091] (g) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 17, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 16; or

[0092] (h) the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 19, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 18.

[0093] In some other embodiments of the application, wherein

[0094] (a) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 5, and the sequence of the light chain variable region is set forth in SEQ ID NO: 4;

[0095] (b) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 7, and the sequence of the light chain variable region is set forth in SEQ ID NO: 6;

[0096] (c) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 9, and the sequence of the light chain variable region is set forth in SEQ ID NO: 8;

[0097] (d) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 11, and the sequence of the light chain variable region is set forth in SEQ ID NO: 10;

[0098] (e) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 13, and the sequence of the light chain variable region is set forth in SEQ ID NO: 12;

[0099] (f) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 15, and the sequence of the light chain variable region is set forth in SEQ ID NO: 14;

[0100] (g) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 17, and the sequence of the light chain variable region is set forth in SEQ ID NO: 16;

[0101] (h) the sequence of the heavy chain variable region is set forth in SEQ ID NO: 19, and the sequence of the light chain variable region is set forth in SEQ ID NO: 18;

[0102] (i) the heavy chain variable region comprises the sequence of SEQ ID NO: 69, and the light chain variable region comprises the sequence of SEQ ID NO: 68; or

[0103] (j) the heavy chain variable region comprises the sequence of SEQ ID NO: 71, and the light chain variable region comprises the sequence of SEQ ID NO: 70.

[0104] In some other embodiments of the application, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and wherein

[0105] (a) the VH comprises a CDRH1, CDRH2, and CDRH3 of a VH as set forth in SEQ ID NO: 5, and / or the VL comprises a CDRL1, CDRL2, and CDRL3 of a VL as set forth in SEQ ID NO: 4;

[0106] (b) the VH comprises a CDRH1, CDRH2, and CDRH3 of a VH as set forth in SEQ ID NO: 7, and / or the VL comprises a CDRL1, CDRL2, and CDRL3 of a VL as set forth in SEQ ID NO: 6;

[0107] (c) the VH comprises a CDRH1, CDRH2, and CDRH3 of a VH as set forth in SEQ ID NO: 9, and / or the VL comprises a CDRL1, CDRL2, and CDRL3 of a VL as set forth in SEQ ID NO: 8;

[0108] (d) the VH comprises a CDRH1, CDRH2, and CDRH3 of a VH as set forth in SEQ ID NO: 11, and / or the VL comprises a CDRL1, CDRL2, and CDRL3 of a VL as set forth in SEQ ID NO: 10;

[0109] (e) the VH comprises CDRH1, CDRH2, and CDRH3 of a VH as shown in SEQ ID NO: 13, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of a VL as shown in SEQ ID NO: 12;

[0110] (f) the VH comprises CDRH1, CDRH2, and CDRH3 of a VH as shown in SEQ ID NO: 15, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of a VL as shown in SEQ ID NO: 14;

[0111] (g) the VH comprises CDRH1, CDRH2, and CDRH3 of a VH as shown in SEQ ID NO: 17, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of a VL as shown in SEQ ID NO: 16;

[0112] (h) the VH comprises CDRH1, CDRH2, and CDRH3 of a VH as shown in SEQ ID NO: 19, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of a VL as shown in SEQ ID NO: 18;

[0113] (i) the VH comprises CDRH1, CDRH2, and CDRH3 of a VH as shown in SEQ ID NO: 69, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of a VL as shown in SEQ ID NO: 68; or

[0114] (j) the VH comprises CDRH1, CDRH2, and CDRH3 of a VH as shown in SEQ ID NO: 71, and / or the VL comprises CDRL1, CDRL2, and CDRL3 of a VL as shown in SEQ ID NO: 70.

[0115] In certain embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof of the present application, (a) some antibodies can block binding of itself (1B2-8C) and Tiragolumab, 4A042-H3, 4A042-H7, and 4B030a, partially block binding of 4A063, but cannot block binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; (b) some antibodies can block binding of itself (4A042-H3), 1B2-8C, 4A042-H7, and 4B030a, without blocking binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; (c) some antibodies can block binding of itself (4A042-H7), 1B2-8C, 4A042-H3, and 4B030a, partially block binding of 4B037a and 4A063, but cannot block binding of 4B056a, 4D035a, and 4E061a; (d) some antibodies can block binding of itself (4B030a), partially block binding of 4B056a and 4A063, and do not block binding of 4B037a, 4D035a, and 4E061a; (e) some antibodies can block binding of themselves (4B037a, 4A063), 4B056a, 4D035a, and 4E061a, and partially block binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a; (f) some antibodies can block binding of itself (4B056a), 4B037a, 4A063, 4D035a, and 4E061a, partially block binding of Tiragolumab, 1B2-8C, 4A042-H3, and 4B030a, but cannot block binding of 4A042-H7; (g) some antibodies can block binding of themselves (4D035a, 4E061a), 4B037a, 4B056a, 4A063, without blocking binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a.

[0116] In some embodiments, the anti-huTIGIT antibodies or antigen-binding fragments thereof of the present application also bind to cynomolgus monkey TIGIT.

[0117] In some other embodiments of the present application, the anti-TIGIT antibodies or antigen-binding fragments thereof further comprise a heavy chain constant region selected from the group consisting of a heavy chain constant region of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0118] In some examples of the present application, the heavy chain constant region is a heavy chain constant region of human IgG or a variant thereof.

[0119] In some other embodiments of the application, the anti-TIGIT antibody or antigen-binding fragment thereof is in a format selected from the group consisting of F(ab')2, Fab', Fab, Fv, scFv, bispecific antibody, and combinations thereof.

[0120] The present application also provides immunoconjugates comprising an anti-TIGIT antibody described herein linked to an agent, such as a detectable label or a cytotoxic agent.

[0121] In other embodiments, the antigen binding domain of the antibody of the present application is present in a bispecific molecule further comprising an antigen binding domain that specifically binds to a different immunomodulatory receptor, including but not limited to PD-1, CTLA-4, or LAG3.

[0122] In a second aspect, the present application provides a polynucleotide encoding an anti-TIGIT antibody or antigen-binding fragment thereof.

[0123] In a third aspect, the present application provides an expression vector expressing the anti-TIGIT antibody or antigen-binding fragment thereof.

[0124] In a fourth aspect, the present application provides an engineered cell comprising a vector expressing the anti-TIGIT antibody or antigen-binding fragment.

[0125] In a fifth aspect, the present application provides a pharmaceutical composition comprising 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, and a pharmaceutically acceptable carrier. The present application 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 antigen-binding fragment thereof, together with instructions for use.

[0126] In a sixth aspect, the present application provides use 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 in the manufacture of a medicament for treating a TIGIT-related disease. Preferably, the TIGIT-related disease is a T cell dysfunction disease; more preferably, the TIGIT-related disease is a tumor, an immunological disease, or an infectious disease; more preferably, the cancer is selected from the group consisting of melanoma, breast cancer, non-small cell lung cancer, colon adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma. More preferably, the cells of the tumor are CD155-positive or PVR-positive.

[0127] In some embodiments, the present application provides a method of enhancing an antigen-specific T cell response, the method comprising contacting a T cell with an anti-huTIGIT antibody or antigen-binding fragment thereof of the present application, such that the antigen-specific T cell response is enhanced, e.g., by reducing an inhibitory signal that would otherwise attenuate an anti-tumor response. In some embodiments, the antigen-specific T cell is a tumor antigen-specific effector T cell, such as a CD8+ T cell, and the increase in anti-tumor activity results, e.g., by blocking TIGIT-mediated inhibitory effects on effector function. Anti-huTIGIT antibodies or antigen-binding fragments thereof of the present application can also reduce inhibitory signals in NK cells, and thus increase their anti-tumor activity. Without intending to be limited by theory, anti-huTIGIT antibodies of the present application increase effector T cell or NK cell function by blocking TIGIT binding to PVR, thereby reducing or eliminating an inhibitory signal that would otherwise be delivered to the cell. Alternatively or in addition, anti-TIGIT antibodies or antigen-binding fragments thereof of the present application can inhibit the interaction between TIGIT and DNAM-1 / CD226, which would otherwise reduce DNAM-1 -mediated immune activation.

[0128] The present application provides a method of reducing or depleting TIGIT+ cells in a tumor in a subject in need thereof, the method comprising administering an effective amount of an anti-huTIGIT antibody of the present application, wherein the antibody has effector function or enhanced effector function to reduce the number of TIGIT+ cells in the tumor. regs The present application provides a method of reducing or depleting TIGIT+ cells in a tumor in a subject in need thereof, the method comprising administering an effective amount of an anti-huTIGIT antibody of the present application, wherein the antibody has effector function or enhanced effector function to reduce the number of TIGIT+ cells in the tumor. regs The present application provides a method of reducing or depleting TIGIT+ cells in a tumor in a subject in need thereof, the method comprising administering an effective amount of an anti-huTIGIT antibody of the present application, wherein the antibody has effector function or enhanced effector function to reduce the number of TIGIT+ cells in the tumor.

[0129] The present application provides a method of enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of an anti-huTIGIT antibody or antigen-binding fragment thereof of the present application, 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 anti-viral immune response is enhanced.

[0130] The present application also provides a method of inhibiting the growth of a tumor in a subject, the method comprising administering to the subject an anti-huTIGIT antibody or antigen-binding fragment thereof of the present application, such that the growth of the tumor is inhibited.

[0131] The present application further provides a method of treating cancer, e.g., by immunotherapy, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-huTIGIT antibody or antigen-binding fragment thereof of the present application, e.g., as a pharmaceutical composition, 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, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer. In certain embodiments, the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

[0132] In a seventh aspect, the present application provides a method of treating a TIGIT- associated disease, comprising administering to a subject in need thereof 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.

[0133] In an eighth aspect, the present application provides a pharmaceutical composition for use in treating a TIGIT-associated 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, or together with, one or more additional therapeutic agents, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG3 antibody, an anti-GITR antibody, an anti-OX40 antibody, an anti-CD73 antibody, an anti-CD40 antibody, an anti-CD137 mAb, an anti-CD27 mAb, an anti-CSF-1R antibody, and / or an anti-CTLA-4 antibody, a TLR agonist, or a small molecule antagonist of IDO or TGF. In particular embodiments, the anti-huTIGIT therapy is combined with an anti-PD-1 and / or anti-PD-L1 therapy, e.g., 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.

[0134] The present application also provides methods of detecting the presence of TIGIT in a sample, on cells within a sample (e.g., FACS) or in a particular location in a cell or tissue (e.g., IHC), or methods of sorting cells based on the presence or absence of TIGIT on their surface (e.g., FACS), comprising contacting a sample with an anti-huTIGIT antibody or antigen-binding fragment thereof of the present application under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof 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.

[0135] The present application uses a transchromosomic mouse (TC-mAb TM mouse) model in which full human antibody gene sequences, including gene regulatory sequences, are transchromosomally transferred. The target antigen is used to immunize the transchromosomic mouse to directly obtain full humanized antibodies. The obtained antibodies do not require subsequent humanization and affinity modification, thereby reducing costs and shortening development cycles. In addition, the antibodies are full human antibodies derived from transchromosomic mice, which significantly reduces their immunogenicity and is more advantageous for drug development. BRIEF DESCRIPTION OF DRAWINGS

[0136] Figure 1 is a graph showing the results of huTIGIT binding affinity using recombinant huTIGIT protein and TC-mAb TM mouse-derived anti-TIGIT antibodies specified in the present application. See Example 3.

[0137] Figure 2A and Figure 2B , 2C shows the results of epitope competition using recombinant huTIGIT protein and TC-mAb TM mouse-derived anti-TIGIT antibodies specified in the present application. See Example 3.

[0138] Figure 3A is a graph showing the results of CHO-TIGIT binding assays at different concentrations using CHO cells expressing human TIGIT and TC-mAb TM mouse-derived anti-TIGIT antibodies specified in the present application.

[0139] Figure 3B is a graph showing the results of CHO-TIGIT binding assays at different concentrations using CHO cells expressing cynomolgus monkey TIGIT (mkTIGIT) and TC-mAb TM mouse-derived anti-TIGIT antibodies specified in the present application. See Example 4.

[0140] Figure 4This is a graph showing the results of CHO-TIGIT CD155 blocking assays using CHO cells expressing human TIGIT and the mouse-derived anti-TIGIT antibody of TC-mAb™ specified in this invention at different concentrations. See Example 5.

[0141] Figure 5A This illustrates the TC-mAb specified in this invention in combination with an anti-PD1 antibody. TM A graph showing the inhibitory effect of mouse-derived anti-TIGIT antibody 4A063 on tumor growth in a human TIGIT transgenic mouse model. Figure 5B The average weight of each group of mice as a function of time is shown. See Example 6.

[0142] Figure 6A This shows the specified TC mAb used in combination with anti-PD1 antibody. TM A graph showing the regulation of the proportion of tumor-infiltrating lymphocytes CD8+ T (CD8+ TIL) within the CD3+ T cell population in CT26 tumors treated with the derived anti-TIGIT antibody 4A063. See Example 7.

[0143] Figure 6B This shows the specified TC mAb used in combination with anti-PD1 antibody. TM A graph showing the regulation of the proportion of CD8+ T cells within the CD3+ T cell population in human TIGIT transgenic mice carrying CT26 tumors treated with the derived anti-TIGIT antibody 4A063. See Example 7. Detailed Implementation

[0144] Definitions

[0145] 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 pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All published materials, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0146] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the disclosure are approximations, 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 testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "1 to 10" is a disclosure of all individual ranges of from 1 to 6.1, from 1 to 5.5, from 1 to 5, from 1 to 4, from 1 to 3.75, from 1 to 3, from 1 to 2.75, from 1 to 2, from 1 to 1.1, from 0 to 0.1, and the like.

[0147] It should also be noted that, as used in this specification and the appended claims, singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "or" is used in the inclusive sense, i.e., the term "or" is used to link a list of items such that any of the items can be used by itself or any combination might be used.

[0148] As used herein, the term "comprising" or "including," or any variation thereof, indicates that the various ingredients can be present together in a mixture or composition of the present application. Thus, the terms "consisting essentially of or "consisting of" are included in the term "comprising" or "including."

[0149] 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 two sequences and refers to the number of identical residues (i.e., amino acids or nucleotides) at positions shared by the compared sequences. Alignment and comparison of sequences can be performed using standard algorithms of the art (e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 101970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444) or by computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI) publicly available in the form of BLAST and FASTA. In addition, ENTREZ available at the National Institutes of Health (Bethesda MD) can be used for sequence comparisons. When using the BLAST or Gapped BLAST programs, the default parameters for each program can be used (e.g., BLASTN, available on the internet website of the National Center for Biotechnology Information). In one embodiment, GCG with a gap weight of 1 can be used to determine 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.

[0150] As used herein, the term "antibody" refers to any antigen binding molecule containing 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, an antibody can contain the Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0151] Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, and epsilon, and define a different class of antibodies, IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgGl, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgMl and IgM2. Thus, "isotype" as used herein means any of the subclasses of immunoglobulin including IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgMl, IgM2, IgD, and IgE. It is understood that therapeutic antibodies can also comprise hybrids of isotypes and / or subclasses.

[0152] As used herein, "CDR region" or "CDR" refers to the hypervariable region 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 an antibody and an antigen or epitope thereof.

[0153] As used herein, the term“antibody fragment” or“antigen-binding fragment” refers to a portion of a full-length antibody and antibody analogs of the antibody that retains the ability to specifically bind with an antigen (e.g., tigit), typically including at least a portion of an antigen binding region or variable region of a parent antibody. In some embodiments, an antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of a light chain). An antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, an antibody fragment retains at least 10% of the parent binding activity when activity is expressed in molar terms. Preferably, an antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% of the binding affinity of the parent antibody to a target. Antibody fragment agents 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), and the like; linear antibodies, single-chain antibodies (e.g., scFv single antibodies) (technology from Genmab), divalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (e.g., VH domain antibodies), domain antibodies (technology from Ablynx); multispecific antibodies (e.g., triabodies, tetrabodies, etc.) formed from antibody fragments; and engineered modified antibodies such as chimeric antibodies (humanized mouse antibodies), heteroconjugate antibodies, and the like. These antibody fragments are obtained using conventional techniques known to those with ordinary skill in the art, and the utility of these fragments is assessed using the same methods as for whole antibodies.

[0154] As used herein, the term“single-chain antibody” refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding with an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0155] In one embodiment, the antibody of the present application can be a multispecific antibody, especially a bispecific antibody, 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 manufactured in a variety of ways known in the art, e.g., prepared chemically or from hybridomas.

[0156] As used herein, the term“transchromosomic mouse (TC-mAb TM mouse)” refers to a mouse containing a mouse artificial chromosome containing a human antibody heavy chain gene or locus and / or a human antibody kappa light chain gene or locus and / or a human antibody lambda light chain gene or locus and at least two mouse 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.

[0157] The antibodies of the present application are typically isolated or recombinant. When "isolated" is used to describe various polypeptides disclosed herein, it is intended to refer to a polypeptide that has been identified and separated and / or recovered from a cell or cell culture from which the polypeptide is expressed. Generally, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities.

[0158] The present application further provides variant antibodies. That is, a number of modifications can be made to the antibodies of the present application, including but 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 differs from that of a parent polypeptide by virtue of at least one amino acid modification. The amino acid modifications can include substitutions, insertions, and deletions. Generally, a variant can include any number of modifications, so long as the function of the protein remains, as described herein.

[0159] As used herein, the term "epitope" refers to a determinant that interacts with a specific antigenic binding site (referred to as a 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 characteristics, as well as specific charge characteristics. A single antigen can have more than one epitope.

[0160] An epitope can include amino acid residues that directly participate in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by the specific antigen binding peptide; in other words, amino acid residues that 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.

[0161] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to an amino acid polymer of any length.

[0162] 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 and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and variants thereof.

[0163] 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 optionally a pharmaceutically acceptable carrier or excipient for accomplishing a particular purpose. In certain embodiments, the pharmaceutical composition comprises a combination that is separated in time and / or space, so long as they are capable of working together to accomplish the purposes of the present disclosure.

[0164] As used herein, "therapeutically effective amount" or "effective amount" means a dosage sufficient to exert a benefit on the subject to which it is administered. The amount actually administered, and the rate and time course of administration, will depend on the condition and severity of the human being treated. Prescription of treatment (e.g., dosage determination, etc.) is ultimately at the discretion of the general practitioner and other medical doctor, who usually considers the disease to be treated, the condition of the individual patient, the delivery site, the method of administration, and other factors known to the doctor.

[0165] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human or non-human, that is provided treatment according to the methods of the present application. Veterinary and non-veterinary applications are contemplated by the present application. The human patient can be an adult or a juvenile (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, hamsters, gerbils, ferrets, rabbits), lagomorphs, swine (e.g., domestic pigs, mini-pigs), equines, canines, felines, bovines, and other domestic, farm, and zoo animals.

[0166] The antibodies and chemotherapeutic agents of the present application are administered to a subject according to known methods, such as intravenous administration in single dose form or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.

[0167] In the present context, the term "pharmaceutically acceptable" means that the compound is physiologically acceptable in that it does not cause adverse effects, such as gastrointestinal disorders, dizziness, or other adverse effects or systemic adverse effects similar to these adverse effects, when administered to a human being.

[0168] 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 lactic acid), 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), and absorption enhancers, flavorings, sweeteners, diluents, coating agents, and the like.

[0169] The term TIGIT or "T-cell immunoreceptor with Ig and ITIM domains" as used herein, unless otherwise indicated, refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), which 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: 1, as well as any form of TIGIT. The term also encompasses naturally occurring variants of TIGIT, e.g., splice variants or allelic variants. The term "TIGIT-associated disease" refers to abnormal expression of TIGIT protein or its ligand CD155 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 an immune-related disease (e.g., T-cell dysfunctional disease) in a subject (e.g., a human). 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 disease, thereby achieving a therapeutic effect on the disease. Such a disease is defined as a TIGIT-associated disease.

[0170] A "T cell dysfunctional disorder" is a T cell disorder or condition characterized by a decreased responsiveness to antigenic stimulation. In some embodiments, a T cell dysfunctional disorder is characterized by T cell exhaustion. In particular embodiments, a T cell dysfunctional disorder is a disorder in which signaling through OX40 and / or OX40L is apparently inadequately reduced. In another embodiment, a T cell dysfunctional disorder is a disorder in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In particular aspects, the decreased responsiveness results in ineffective control of a pathogen or tumor expressing an immunogen. Examples of T cell dysfunctional disorders characterized by T cell dysfunction include unresolved acute infection, chronic infection, and tumor immunity. In some embodiments, the subject is a human.

[0171] The terms "cancer" and "tumor" are used interchangeably. They refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can result in the formation of malignant tumors or cells invading neighboring tissues, and can spread through the lymphatic system or bloodstream to distant parts of the body. Cancers include benign and malignant cancers and dormant tumors or micrometastases. Cancers also include hematological malignancies.

[0172] "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's lymphoma and the majority of non-Hodgkin's 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 (overlaps with chronic lymphocytic leukemia), Burkitt's lymphoma, mantle cell lymphoma (MCL), mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal 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 myelogenous leukemia, chronic myelogenous 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.

[0173] The term "PVR-positive tumor" refers to a tumor in which the expression of PVR is increased in the cancer tissue. PVR-positive tumors include, but are not limited to, adrenocortical carcinoma, chromophobe renal cell carcinoma, papillary renal carcinoma, hepatocellular carcinoma of the liver, pancreatic ductal adenocarcinoma, pheochromocytoma and paraganglioma, lung adenocarcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, uterine corpus endometrial carcinoma, cervical cancer, cutaneous melanoma, mesothelioma, urothelial bladder carcinoma, colon and rectum adenocarcinoma, clear cell kidney cancer, lung squamous cell carcinoma, uterine carcinosarcoma, sarcoma, ovarian serous cystadenocarcinoma, papillary thyroid carcinoma, glioblastoma multiforme, breast carcinoma, low-grade glioma, and diffuse B-cell lymphoma.

[0174] In the present context, the term "immune-related disease" refers to an immune-related disease in a mammal caused by, mediated by, or otherwise contributed to by components of the mammal's immune system, and also includes diseases that stimulate or interfere with the immune response such that disease progression can be improved. "Immune-related diseases" include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumors, and the like.

[0175] The anti-TIGIT antibodies or antigen-binding fragments thereof of the present application can be used to treat an infection or infectious disease in a subject (e.g., a human). In some preferred embodiment options, the infection or infectious disease is selected from the group consisting of viral infection, bacterial infection, fungal infection, and parasitic infection, including but not limited to HIV, hepatitis virus, herpes virus, CMV, EBV, influenza.

[0176] In the following, some preferred embodiments and aspects of the present application will be further described in connection with specific examples, and these examples are not to be construed as limiting the scope of the present application.

[0177] Example

[0178] Example 1 Generation of anti-TIGIT monoclonal antibodies

[0179] 1. TIGIT recombinant proteins for antigen immunization and binding assays

[0180] A cDNA encoding full-length human TIGIT (huTIGIT, SEQ ID NO: 1) based on GenBank sequence (Locus: NM_173799) was synthesized and purchased from Eurofins. A coding region for extracellular domain ECD corresponding to amino acid (AA) 1-141 of full-length human TIGIT (SEQ ID NO: 2) was amplified by PCR and cloned 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, the recombinant fusion protein (Trx-TIGIT-HIS and Gst-TIGIT-HIS) expression plasmids were transferred to competent E. coli (E. coli gamiB (DE3) pLysS' Novagen) and cultured. After IPTG induction, the E. coli cells were centrifuged to collect the precipitates. After sonicating the E. coli, the precipitates were obtained by centrifugation. A solubilization reagent was added to dissolve the precipitates, which were then purified by a Ni-NTA column (Qiagen, Ni-NTA Superflow, #30410) and dialyzed. The purified recombinant proteins were detected by PAGE, and then the recombinant proteins were stored in small aliquots at -30°C.

[0181] ATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACCTCAGGAGAAAATCAGCTGGACAGGAGGAATGGAGCCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAGAGACTGGTTAG (SEQ ID NO: 1);

[0182] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAI CNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTY TGRIFLEVLESSVAEHGAR FQIP (SEQ ID NO: 2).

[0183] 2. Establishment of stable expression cell lines

[0184] cDNAs encoding full-length human TIGIT (huTIGIT, SEQ ID NO: 1) and cynomolgus monkey TIGIT (mkTIGIT, SEQ ID NO: 3) based on GenBank sequences (NM_73799) and (XM_005548101.2), respectively, were synthesized and purchased from Genscript. After PCR amplification, the DNA products were cloned into pcDNA3.1 expression vector (Invitrogen) and transduced into CHO-K1 cell line (JCRB, #JCRB9018) to generate CHO-huTIGIT and CHO-mkTIGIT cell lines. HuTIGIT or mkTIGIT high expression stable cell lines were selected by culturing in medium with G418, eGFP expression and FACS binding assay.

[0185] ATGCGGTGGTGTCTCTTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAAAGAAAGGTGGCTCTGTTATCTTACAATGTCACCTCTCCTCCACCATGGCACAAGTGACCCAGGTCAACTGGGAGCAGCATGACCATTCGCTTCTGGCCATTCGTAATGCTGAGTTGGGGTGGCACATCTACCCAGCCTTCAAGGATCGAGTGGCCCCGGGTCCTGGCCTGGGCCTCACCCTCCAGTCGCTGACCATGAATGATACAGGGGAGTACTTCTGCACCTATCACACCTACCCTGATGGGACTTACAGAGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACAGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCATGATGCTGGTGGTCATCTGCATAGCAGTCATCGTGGTGGTCGTGTTGGCTAGAAAGAAGAAATCCCTCAGAATCCATTCTGTGGAAAGTGGCCTCCAGAGAAAATCAACTGGACAGGAAGAACAGATTCCCAGTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCTGGGCTCTGTGGAGAGCAGCAGGGAGATGACTGTGCCGAGCTGCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGGAGCTGCAGCTTCTTCACAGAGACTGGGTAG (SEQ ID NO:3).

[0186] 3. Immunization, hybridoma fusion and cloning

[0187] Human TIGIT recombinant protein (Trx-huTIGIT-HIS fusion protein, 100 pg / mouse / primary, 50 pg / mouse / boost) was mixed with Freund's complete adjuvant (FCA, purchased from BD, Cat# 263810, 100 pL / mouse / primary) for primary immunization injection of 6-8 weeks old transchromosomic mice (TC-mAb TM mice), and Freund's incomplete adjuvant (FIA, purchased from Sigma, Cat# F5882, 100 pL / mouse / boost) was used for boost injection. SAS, purchased from sigma, cat# s6322-1vl, 50 μL / mouse / boost) for boost immunization injection, with 2-3 weeks interval. The last immunization does not require adjuvant, only Trx-huTIGIT-HIS fusion protein (50 μg / mouse / final). All immunizations were performed by intraperitoneal injection.

[0188] Three days after the last immunization, mice were euthanized, spleen and lymph nodes were removed aseptically, 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 96-well plates, incubated at 37 °C and 5% CO2 for 7 days, replaced with HT medium, and incubated for 5 days.

[0189] 4. Evaluation of the binding activity of anti-TIGIT antibodies by ELISA, immunocytochemistry (ICC) and flow cytometry

[0190] ELISA using human TIGIT protein. 96 well plates (Nunc, Cat# 44-2404) were coated with Gst-huTIGIT-HIS fusion protein and Trx-huTIGIT-HIS (50 ng / well) respectively, diluted with PBS buffer, and incubated at 4°C overnight. Then the wells were 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 were added and incubated at room temperature. To detect the specificity of the antibodies, horseradish peroxidase-conjugated anti-human IgG antibody (goat anti-human IgG-Fc fragment cross-adsorbed antibody HRP-conjugated, BETHYL, #A80-304P) was diluted to the optimal concentration in PBS containing 0.05% Tween-20, then added at 100 μL / well after washing, incubated at room temperature for 0.5 hours. The plates were washed 3 times using 300 μL of TBS containing 0.05% Tween-20. 100 μL of substrate solution containing 0.5 mg / mL OPD and 0.03% H2O2 was added, the plates were incubated at room temperature for 30 minutes, then 25 μL of 1M H2SO4 (Nacalai Tesque, #95626-06) was added, and then read at 492 nm. Positive well clones were selected and inoculated into new 96 well plates. After 3 days, supernatants of the new 96 well plates were screened by ELISA using human TIGIT protein. Hybridoma cell lines that bound human TIGIT were expanded and cultured for 2-4 days, ELISA positive clones were detected by ICC and flow cytometry using CHO-huTIGIT and CHO-mkTIGIT cells, and positive clones were selected. After several days of culture, a secondary detection was performed according to the above method. Secondary positive clones were diluted to the limit, and after two weeks, ICC and flow cytometry were tested again, and limit dilution was performed again.

[0191] Example 2 Sequence analysis of anti-TIGIT antibodies

[0192] After initial screening by ELISA, ICC and FACS, positive hybridoma clones were subcloned by limiting dilution. After re-verification, cloned hybridoma cells were cultured in 10 cm culture dishes. When the cell density reached 80 to 90%, the cells were collected and suspended with solution. RNA was extracted from the suspended cells with microKit (QIAGEN #74104). 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. According to the sequence, TIGIT antibody expression plasmids were constructed, and expressed in HEK293 cells. Eight antibodies were purified by protein A, and the antibody purity was over 95% by SDS-PAGE analysis. The obtained ten 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.

[0193] Table 1 TC mAbs in the present invention TM Variable region sequences of VH region and VL region of mouse-derived anti-TIGIT antibodies

[0194]

[0195]

[0196]

[0197]

[0198] CDRs were predicted for VL and VH using IMGT program, and the results are shown in Table 2.

[0199] Table 2 Predicted CDRs of eight antibodies

[0200]

[0201]

[0202] It should be noted that when different CDR prediction programs are used, the CDRs of the same VH or the same VL can 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.

[0203] Example 3 Designated TC-mAbs TM Determination of affinity between mouse-derived anti-TIGIT antibodies and recombinant human TIGIT protein

[0204] The equilibrium dissociation constants (KD) of the eight example antibodies mentioned above in this invention that bind to human TIGIT were measured using a FortebioOctet RED96. The measurement method was performed according to existing methods (Estep, Petal., High throughput solution Based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2):p.270-8). For the TC-mAbs specified in this invention... TM The affinity between mouse-derived anti-TIGIT antibody and TIGIT-HIS (Biointron, BI120) was measured. An NTA (HIS-tag) sensor was used. After equilibration of the sensor in analysis buffer, human TIGIT-HIS was loaded onto the NTA sensor (fortebio) for affinity measurement. The antigen-loaded sensor was placed in solutions containing antibodies (antibody concentrations of 5, 2.5, 0.83, 0.278, 0.09, 0.03, and 0.01 μg / ml) until the plateau phase, and then the sensor was transferred to analysis buffer for dissociation for at least 2 minutes to measure the dissociation rate. A 1:1 combinatorial model was used for dynamic analysis.

[0205] In the experiments described above, the TC-mAb specified in this invention... TM The KD values ​​of mouse-derived anti-TIGIT antibodies are shown in Table 3.

[0206] Table 3. Unit valence (KD) of eight antibodies

[0207]

[0208] The epitope of binding to human TIGIT of all human TIGIT antibodies (1B2-8C, 4A042-H3, 4A042-H7, 4B030a, 4B037a, 4B056a, 4A063, 4D035a, 4E061a) was investigated using Octet binding test compared to the anti-TIGIT reference antibody Tiragolumab (synthesized according to Tiragolumab sequence disclosed in KEGG-DRUG database). The experiment was performed as follows: Sensors loaded with TIGIT-HIS (Biointron, BI120) were placed in solutions containing TIGIT antibodies until plateau, then the sensors were transferred into analysis buffer to saturation, then the sensors were transferred into other analytes containing the reference antibody (Tiragolumab) or buffer. After binding to plateau, it was eluted. Epitope grouping shows that there is competition between three candidate clones epitopes compared to Tiragolumab, that is to say, they combine with the same antigenic epitope of TIGIT. The results are shown in Figure 2A

[0209] ​Epitope competition exists between the various antibodies of the present invention. 1B2-8C can block binding of itself and Tiragolumab, 4A042-H3, 4A042-H7 and 4B030a, partially block binding of 4A063, but does not block binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a; Tiragolumab and 4A042-H3 can block binding of Tiragolumab, 1B2-8C, 4A042-H7 and 4B030a, but does not block binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a; 4A042-H7 can block binding of itself and Tiragolumab, 1B2-8C, 4A042-H3 and 4B030a, partially block binding of 4B037a and 4A063, but does not block binding of 4B056a, 4D035a and 4E061a; 4B030a can block binding of itself, partially block binding of 4B056a and 4A063, and does not block binding of 4B037a, 4D035a and 4E061a; 4B037a and 4A063 can block binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a, and partially block binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7 and 4B030a; 4B056a can block binding of itself and 4B037a, 4A063, 4D035a and 4E061a, partially block binding of Tiragolumab, 1B2-8C, 4A042-H3 and 4B030a, but does not block binding of 4A042-H7; 4D035a and 4E061a can block binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a, but does not block binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7 and 4B030a. The results are shown in Figure 2B and 2C .

[0210] TC-mAbs specified in Example 4 TM Binding activity of mouse-derived anti-TIGIT monoclonal antibodies to cell surface TIGIT

[0211] CHO-huTIGIT cells or CHO-mkTIGIT cells were implanted in 96-well plates. The antibodies of Example 2 were diluted to different concentrations, respectively, and added to the 96-well plates covered with cells (100 μL / well), incubated at 4°C (on ice) for 1 hour.

[0212] Cells were washed with 200 μL of wash buffer per well and centrifuged at 1600 rpm (approximately 260 × g) at 4°C for 3 minutes, discarding the supernatant. This process was repeated twice. 30 μL of anti-human IgG secondary antibody (Jackson Immuno Research, #109-585-190, Alexa) was added to each well. Cells were washed with 594 AffiniPure goat anti-human IgG (Fcγ fragment specific) wash buffer and incubated at 4°C (on ice) for 1 hour. After washing twice, cells were transferred to flat-bottomed 96-well plates and analyzed using CytoFLEX S. Antibody concentration was plotted on the logarithm of 10 as the x-axis and the median fluorescence value of the two channels as the y-axis. EC 50 (CHO-huTIGIT and CHO-mkTIGIT) were compared with the peak values ​​of the curves. (μg / mL) results are shown in Table 4 and Figures 3A-3B middle.

[0213] Table 4. Binding activity of antibodies to human TIGIT or cynomolgus monkey TIGIT (EC) 50 (μg / mL)

[0214]

[0215]

[0216] Example 5: TC-mAb in this invention TM Mouse-derived anti-TIGIT monoclonal antibodies block the binding of TIGIT to CD155.

[0217] CHO-huTIGIT-eGFP cells were implanted into V-bottom 96-well plates. The antibody from Example 2 and the reference antibody were diluted to different concentrations and added (100 μL / well) to 96-well plates covered with cells (100%), and incubated at 4°C for 1 hour.

[0218] Cells were washed by adding 200 pL / well of wash buffer and centrifuged at 1600 rpm (about 260 x g) for 3 minutes at 4°C and the supernatant was discarded, repeated twice. 30 pL of wash buffer containing biotinylated human CD155 (human CD155 / PVR / NECL5 protein (Fc tag), biotinylated, Sin Biological, 10109-H02H-B) was added, then the cells were incubated at 4°C (on ice) for 1 hour. 30 pL of wash buffer containing streptavidin-594 (f.c. = 10 pg / ml) was added to the wells, then the cells were incubated at 4°C for 30 minutes. Washed once, and the cells were transferred to a flat-bottom 96-well plate and analyzed with CytoFLEX S. The antibody concentration (pg / mL) was used as the abscissa with a base of 10, and the median fluorescence value corresponding to each antibody concentration was used as the ordinate. The blocking ability of different antibodies to block the binding between CD155 and TIGIT was distinguished by analyzing the IC 50 of the curve.

[0219] Eight antibodies were tested for their blocking ability to CD155 using IgG1 as a negative control antibody. The IC 50 ( pg / mL) results are shown in Table 5 and Figure 4 .

[0220] Table 5 TC-mAb TM specified in Table 5. 50

[0221]

[0222]

[0223] Example 6 In vivo efficacy of combination therapy of anti-TIGIT antibodies and anti-PD-1 antibodies

[0224] To evaluate the synergistic effect of anti-TIGIT antibodies and anti-PD-1 antibodies in vivo, the mouse colorectal cancer cell line CT26.WT (5 x 10 5 cells / mouse) was transplanted subcutaneously into TIGIT humanized BALB / c mice. When the average tumor volume reached 120 ± 50 mm 3 ​At day 0, mice were inoculated subcutaneously with 5 x 105MC38 cells in 50 μL of PBS. At day 3, mice were randomized into groups (n=3) according to tumor volume and administered IgG negative control antibody (anti-HEL human IgGl isotype, biointron, 200 μg / mouse / treatment), anti-mPD-1 antibody (InVivoMAb anti-mouse PD-1, lot: 795720D1, 20 μg / mouse / treatment), anti-mPD-1 antibody (20 μg / mouse / treatment) in combination with positive reference antibody (Tiragolumab-hIgGl, 200 μg / mouse / treatment), anti-mPD-1 antibody (20 μg / mouse / treatment) in combination with 4A063 antibody (4A063-hIgGl, 200 μg / mouse / treatment), once every three days for a total of 6 treatments. Tumor volume and body weight were measured twice a week.

[0225] Results are shown in Figure 5A and Figure 5B which provide the mean tumor volume of each group of mice as a function of time and the mean weight of each group of mice as a function of time. 4A063 exhibited a significant inhibition of tumor growth (TGI: 44% vs 92.33%) compared to the positive reference antibody and there was no significant difference in mean body weight between groups.

[0226] Characterization of the mechanism of action of the anti-tumor activity of the combination therapy of anti-TIGIT antibodies and anti-PD-1 antibodies in vivo

[0227] To investigate the in vivo mode of action of anti-TIGIT antibodies, the immune cell infiltration of tumors was analyzed by flow cytometry after combination treatment with anti-TIGIT antibody 4A063 (hIgGl) and anti-PD-1 antibody. Mice were inoculated and treated as described in Example 6. Three days after the six treatments, 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 spleen single cell suspensions were obtained directly after spleen grinding. After staining with Fc-block, 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 commercial buffer (BD Cytofix / Cytoperm Fixation / Permeabilization Kit, 554714), cells were stained with anti-IFNy antibody (PerCP / Cyanine5.5 anti-mouse IFN-g, Biolegend, 505822). After routine washes and filtration, cells were analyzed by flow cytometry. TM Results are shown in

[0228] Results are shown in Figure 6A andFigure 6B In summary. Figure 6A It was shown that treatment of tumors in vivo with the anti-TIGIT antibody 4A063 hIgGl resulted in an increased proportion of CD8+ TIL population in the tumor microenvironment of the combination treatment (P=0.0335) compared to the control group, which was not observed in the single PD-1 antibody group. At the same time, the flow cytometry results of T cells in the spleen (which are shown in Figure 6B Summary In summary + The increase of CD8+ TIL was more pronounced in the 4A063-hIgGl combination group compared to the anti-TIGIT reference antibody (Tiragolumab-hIgGl) combination group. For the PD-1 antibody in combination with the anti-TIGIT antibody 4A063 hIgGl, the function of T cells within the tumor was also improved and the CD3+ CD8+ T cells produced increased amounts of IFNy.

[0229] All publications and patents cited in this application are incorporated by reference. Various modifications and variations to the described methods and compositions of the application will be apparent to those skilled in the art without departing from the scope and spirit of the application. Although the application has been described in connection with specific preferred embodiments, it should be understood that the application as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the application that are obvious to those skilled in the relevant fields are intended to be within the scope of the claims.

Claims

1. An anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region has amino acid sequences CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, and the light chain variable region has amino acid sequences CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NO:56, GAS, and SEQ ID NO:58, respectively.

2. The anti-TIGIT antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

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

4. The anti-TIGIT antibody or its antigen-binding fragment 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 its antigen-binding fragment according to claim 1 or 2, wherein the anti-TIGIT antibody or its antigen-binding fragment is selected from the group consisting of: F(ab')2, Fab', Fab, Fv, scFv and combinations thereof.

6. The anti-TIGIT antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The anti-TIGIT antibody or its antigen-binding fragment is conjugated with a reagent, wherein the reagent is a detectable label.

7. A polynucleotide encoding an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An expression vector capable of expressing an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

9. An engineered cell comprising the polynucleotide of claim 7 or the expression vector of claim 8.

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

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

12. The pharmaceutical composition according to claim 11, wherein, The other 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 other immune checkpoint inhibitor is an antibody or its antigen-binding fragment that targets another immune checkpoint.

14. Use of the anti-TIGIT antibody or 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 to 13 for the manufacture of a medicament for the treatment of TIGIT-related diseases; The TIGIT-related diseases mentioned are tumors, among which, The tumor cells are CD155 positive or PVR positive; and The tumors are selected from the group consisting of: melanoma, breast cancer, non-small cell lung cancer, colon adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma.

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