Tigit antibodies and uses thereof

Abstract

The invention provides an anti-TIGIT antibody specifically bound with TIGIT or an antigen binding fragment thereof, a composition containing the anti-TIGIT antibody or the antigen binding fragment thereof, and application of the anti-TIGIT antibody or the antigen binding fragment thereof in diagnosis and treatment of TIGIT related diseases.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 2022800819862 and invention name “TIGIT antibodies and their uses”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to PCT application No. PCT / CN2021 / 139122, filed on December 17, 2021, entitled “TIGIT Antibodies and Uses Thereof,” the disclosure of which is incorporated herein by reference. Technical Field

[0004] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to TIGIT (T cell immunoreceptor having Ig and ITIM domains), pharmaceutical compositions containing anti-TIGIT antibodies or antigen-binding fragments thereof, and uses thereof. Background Art

[0005] In recent years, tumor immunotherapy has achieved tremendous breakthroughs and has become a new hope in cancer treatment. In particular, therapies that block tumor immunosuppressive checkpoints, such as PD-1 / PD-L1 and CTLA-4, have attracted considerable attention. Since 2000, the FDA has approved PD-1 / PD-L1 monoclonal antibodies for the clinical treatment of malignancies such as melanoma, non-small cell lung cancer, and prostate cancer, with promising therapeutic effects. However, the clinical response rate of PD-1 / PD-L1 monoclonal antibody therapy remains limited, which significantly limits its clinical application. Therefore, the search for new immunosuppressive checkpoints has become a research hotspot.

[0006] TIGIT is a new immunosuppressive 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, which include CD4 + T cells, CD8 +T cells and Treg cells. Under normal conditions, TIGIT expression is low, but when T cells and NK cells are activated, TIGIT expression increases significantly (J Immunol, 2012, 188:3869-3875, Cancer Cell 26, 923–937, Nat Immunol, 19, 723–732). Currently, the discovered TIGIT ligands include CD155, CD112, and CD113, of which CD155 is the primary ligand for TIGIT. Crystal structure analysis shows that TIGIT and CD155 form homodimers, 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, macrophages, and non-hematopoietic tissues (such as the kidney, nervous system, and small intestine). Similar to TIGIT, the activating receptors DNAM-1 and CD96 can also bind to CD155, but their affinity is weaker than that of TIGIT. In summary, the ligand-receptor interaction mode of TIGIT / CD155 is similar to the CTLA-4 / CD28 pathway. Inhibitory receptors with high affinity and activating receptors with low affinity compete for binding to the same ligand, thereby precisely regulating the immune response. TIGIT bound to CD155 can exert immunosuppressive effects by regulating DC function, inhibiting effector T cell activity, interfering with DNAM-1 coactivation, and increasing Treg inhibition (Clinical and Experimental Immunology, 2020 May; 200(2):108-119, Immunity 40, 569–581).

[0007] Several studies based on humans and mice have shown 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 + Highly expressed in T cells, tumor-infiltrating Tregs and NK cells. +TIGIT expression in T cells and NK cells is usually 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 in malignant tumors. High expression of TIGIT in NK cells is associated with the severity of the disease. TIGIT knockout mice have significantly reduced tumor growth and increased survival rate.

[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 no hematopoietic cell development disorders. The incidence of autoimmune diseases increases only after crossing with mice with a tendency to autoimmune diseases. Compared with PD-1 and CTLA-4 mAbs, animal experiments have shown that the incidence of irAEs during the administration of anti-TIGIT mAbs is lower (Oncoimmunology 2018; 7: e1445949). Therefore, antibody drugs targeting TIGIT have a relatively low risk of side effects and are high-quality candidate targets for clinical anti-cancer drugs.

[0009] Published clinical trial results have shown that the combination of TIGIT antibodies and PD-1 / PD-L1 monoclonal antibodies can significantly increase patient response rates, improve treatment outcomes, and address drug resistance in some patients (Cancers 2019; 11:877, Cancer Discov, 10:1086-1087 (2020)). Currently, no monoclonal antibodies targeting TIGIT have been approved for marketing worldwide, so it is necessary to develop antibodies with high affinity and activity as candidate drugs. Summary of the Invention

[0010] After extensive experiments, the inventors of the present invention used transchromosomal mice (TC mAb) that have all human antibody gene sequences. TM mice) platform for screening and unexpectedly obtained antibodies that specifically bind to TIGIT, which showed excellent affinity for TIGIT and had potential drug development prospects.

[0011] The present invention provides improved drugs and treatments 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 properties, such as high-affinity specific binding to huTIGIT, binding to monkey TIGIT (e.g., cynomolgus monkey TIGIT), the ability to block the binding of TIGIT to PVR and Nectin-2, the ability to block the interaction of TIGIT with DNAM, or any combination of these properties.

[0012] The present invention relates to antibodies that compete with and cross-block the binding of antibodies having the heavy and light chain variable domain sequences disclosed herein 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 co-stimulation of NK cells to increase NK-mediated anti-tumor response killing. In another embodiment, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention deplete regulatory T cell populations within the tumor, which would otherwise inhibit the anti-tumor immune response. In another embodiment, the anti-TIGIT antibodies of the present invention in the form of IgG1 deplete CD8+ exhausted T cells and Tregs, thereby allowing the influx of fresh non-exhausted CD8+ T cells. In some embodiments, the anti-TIGIT antibodies of the present invention in the form of IgG1 increase the proportion of CD8+ TIL populations 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 Fcγ receptors (FcγRs), for example, in embodiments that rely on enhancing the 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, for example, 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 invention provides an anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to TIGIT. The anti-TIGIT antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises CDRL1, CDRL2, and CDRL3.

[0016] In some embodiments of the present invention, 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 adding, deleting or substituting 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 the 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 the 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 adding, deleting or substituting one or more amino acids.

[0024] In some other embodiments of the present invention, 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 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: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 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: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 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: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 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: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 present invention, wherein

[0032] (a) the CDRH1 comprises a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, SEQ ID NO:75, and SEQ ID NO:81;

[0033] (b) the CDRH2 comprises a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:76, and SEQ ID NO:82;

[0034] (c) the CDRH3 comprises a sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:77, and SEQ ID NO:83;

[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 present invention, wherein the heavy chain variable region comprises CDRH1, CDRH2 and CDRH3, and wherein

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

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

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

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

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

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

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

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

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

[0048] (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.

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

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

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

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

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

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

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

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

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

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

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

[0060] In some other embodiments of the present invention, wherein

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

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

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

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

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

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

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

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

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

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

[0074] (c) the sequence of CDRH1 is shown in SEQ ID NO:35, the sequence of CDRH2 is shown in SEQ ID NO:36, and the sequence of CDRH3 is shown in SEQ ID NO:37; and the sequence of CDRL1 is shown in SEQ ID NO:32, the sequence of CDRL2 is shown in SEQ ID NO:33, and the sequence of CDRL3 is shown 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 shown in SEQ ID NO:47, the sequence of CDRH2 is shown in SEQ ID NO:48, and the sequence of CDRH3 is shown in SEQ ID NO:49; and the sequence of CDRL1 is shown in SEQ ID NO:44, the sequence of CDRL2 is shown in SEQ ID NO:45, and the sequence of CDRL3 is shown 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 shown in SEQ ID NO:59, the sequence of CDRH2 is shown in SEQ ID NO:60, and the sequence of CDRH3 is shown in SEQ ID NO:61; and the sequence of CDRL1 is shown in SEQ ID NO:56, the sequence of CDRL2 is shown in SEQ ID NO:57, and the sequence of CDRL3 is shown 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 shown in SEQ ID NO:81, the sequence of CDRH2 is shown in SEQ ID NO:82, and the sequence of CDRH3 is shown in SEQ ID NO:83; and the sequence of CDRL1 is shown in SEQ ID NO:78, the sequence of CDRL2 is shown in SEQ ID NO:79, and the sequence of CDRL3 is shown in SEQ ID NO:80.

[0082] In some other embodiments of the present invention, 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 present invention, 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 present invention, wherein

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

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

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

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

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

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

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

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

[0093] In some other embodiments of the present invention, wherein

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

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

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

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

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

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

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

[0101] (h) the sequence of the heavy chain variable region is shown in SEQ ID NO: 19, and the sequence of the light chain variable region is shown 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 present invention, 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 CDRH1, CDRH2 and CDRH3 of the VH as shown in SEQ ID NO: 5, and / or the VL comprises CDRL1, CDRL2 and CDRL3 of the VL as shown in SEQ ID NO: 4;

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

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

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

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

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

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

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

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

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

[0115] In certain embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof of the present invention, (a) some antibodies can block the binding of themselves (1B2-8C) and Tiragolumab, 4A042-H3, 4A042-H7 and 4B030a, partially block the binding of 4A063, but cannot block the binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a; (b) some antibodies can block the binding of themselves (4A042-H3), 1B2-8C, 4A042-H7 and 4B030a, partially block the binding of 4A063, but cannot block the binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a; (c) Some antibodies can block the binding of themselves (4A042-H7), 1B2-8C, 4A042-H3 and 4B030a, partially block the binding of 4B037a and 4A063, but cannot block the binding of 4B056a, 4D035a and 4E061a; (d) Some antibodies can block the binding of themselves (4B030a), partially block the binding of (e) Some antibodies can block the binding of themselves (4B037a, 4A063), 4B056a, 4D035a, and 4E061a, and partially block the binding of tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a; (f) Some antibodies can block the binding of themselves (4B056a), 4B037a, 4A063, 4B056a, 4D035a, and 4E061a, and partially block the binding of tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a. 063, 4D035a and 4E061a, and partially blocked the binding of Tiragolumab, 1B2-8C, 4A042-H3 and 4B030a, but could not block the binding of 4A042-H7; (g) Some antibodies could block the binding of themselves (4D035a, 4E061a), 4B037a, 4B056a, 4A063, but did not block the 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 invention also bind to cynomolgus monkey TIGIT.

[0117] In some other embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

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

[0120] The invention 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 an antibody of the invention 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 invention provides a polynucleotide encoding an anti-TIGIT antibody or an antigen-binding fragment thereof.

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

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

[0125] In a fifth aspect, the present invention 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 invention further provides an antibody-drug conjugate comprising the anti-TIGIT antibody or antigen-binding fragment thereof of the first aspect. Also provided herein is a kit containing the anti-TIGIT antibody or antigen-binding fragment thereof and instructions for use.

[0126] In a sixth aspect, the present invention provides the 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 immune disease, or an infectious disease; more preferably, the cancer is selected from the group consisting of: melanoma, breast cancer, non-small cell lung cancer, colon adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma. More preferably, the tumor cells are CD155-positive or PVR-positive.

[0127] In some embodiments, the present invention provides a method for 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 invention, such that the antigen-specific T cell response is enhanced, for example, by reducing inhibitory signals that would otherwise attenuate the 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 anti-tumor activity is increased, for example, by blocking the enhancement of the inhibitory effect mediated by TIGIT. The anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention can also reduce inhibitory signals in NK cells and thereby increase their anti-tumor activity. Without intending to be limited by theory, the anti-huTIGIT antibody of the present invention increases effector T cell or NK cell function by blocking the binding of TIGIT to PVR, thereby reducing or eliminating inhibitory signals that would otherwise be delivered to the cell. Alternatively or in addition, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention can inhibit the interaction between TIGIT and DNAM-1 / CD226, which would otherwise reduce DNAM-1-mediated immune activation.

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

[0129] The present invention provides a method for enhancing an immune response in a subject, comprising administering to the subject an effective amount of an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention, such that the immune response in the subject is enhanced. In certain embodiments, the subject has a tumor, and the immune response against the tumor is enhanced. In another embodiment, the subject has a viral infection, and the antiviral immune response is enhanced.

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

[0131] The present invention further provides a method for treating cancer, for example, by immunotherapy, comprising administering a therapeutically effective amount of an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention, for example as a pharmaceutical composition, to a subject in need thereof, thereby treating the cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasms, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancers. In certain embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

[0132] In the seventh aspect, the present invention provides a method for treating TIGIT-related diseases, comprising administering an effective amount of the anti-TIGIT antibody or antigen-binding fragment thereof of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, the cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect to a subject in need thereof.

[0133] In an eighth aspect, the present invention provides a pharmaceutical composition for treating a TIGIT-related disease, wherein the pharmaceutical composition comprises the anti-TIGIT antibody or antigen-binding fragment thereof of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, or the cell of the fourth aspect. In combination with one or more additional therapeutic agents, or as a bispecific agent together with one or more additional therapeutic agents, the therapeutic agent is, for example, 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-CD27mAb, an anti-CSF-1R antibody and / or an anti-CTLA-4 antibody, a TLR agonist, or a small molecule antagonist of IDO or TGFβ. In a specific embodiment, anti-huTIGIT therapy is combined with 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 invention also provides a method for detecting the presence of TIGIT in a sample, on cells within a sample (e.g., FACS), or in a specific location in a cell or tissue (e.g., IHC), or a method for sorting cells based on the presence or absence of TIGIT on their surface (e.g., FACS), the method comprising contacting the sample with an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention under conditions that allow 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 invention uses transchromosomal mice (TC-mAb) into which fully human antibody gene sequences (including gene regulatory sequences) are transferred. TM The target antigen is used to immunize transgenic mice to directly obtain fully humanized antibodies. The obtained antibodies do not require subsequent humanization and affinity modification, thereby reducing costs and shortening the development cycle. In addition, the antibodies are fully human antibodies derived from transgenic mice, which significantly reduces their immunogenicity and is more conducive to drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Figure 1 The present invention shows the use of recombinant huTIGIT protein and the TC-mAb specified in the present invention. TM Graph of results showing huTIGIT binding affinity of mouse-derived anti-TIGIT antibodies. See Example 3.

[0137] Figure 2A and Figure 2B 、 2C The use of recombinant huTIGIT protein and the TC-mAb specified in the present invention is shown. TM Results of epitope competition with mouse-derived anti-TIGIT antibodies. See Example 3.

[0138] Figure 3A Figure 2 shows the use of CHO cells expressing human TIGIT and the TC-mAb specified in the present invention. TM Graph of the results of a CHO-TIGIT binding assay with mouse-derived anti-TIGIT antibodies at various concentrations.

[0139] Figure 3B Figure 2 shows the use of CHO cells expressing cynomolgus monkey TIGIT (mkTIGIT) and the TC-mAb specified in the present invention. TM Graph of the results of a CHO-TIGIT binding assay with mouse-derived anti-TIGIT antibodies at various concentrations. See Example 4.

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

[0141] Figure 5A The TC-mAb specified in the present invention in combination with anti-PD1 antibody is shown. TM Graph showing the inhibitory effect of the mouse-derived anti-TIGIT antibody 4A063 on tumor growth in a human TIGIT transgenic mouse model. Figure 5B Shown are the mean body weights of mice in each group as a function of time. See Example 6.

[0142] Figure 6A Figures show the indicated TC mAbs in combination with anti-PD1 antibodies. TM Figure 2 shows the modulation of the proportion of tumor-infiltrating lymphocytes (CD8+ TILs) within the CD3+ T cell population in CT26 tumors treated with the derived anti-TIGIT antibody 4A063. See Example 7.

[0143] Figure 6B Figures show the indicated TC mAbs in combination with anti-PD1 antibodies. TM Graph showing the modulation of the proportion of CD8+ T cells within the CD3+ T cell population in splenocytes from human TIGIT transgenic mice bearing CT26 tumors treated with the derived anti-TIGIT antibody 4A063. See Example 7. DETAILED DESCRIPTION

[0144] definition

[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 the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0146] Although numerical ranges and parameter approximations are shown in the broad scope of the present disclosure, the numerical values ​​shown in the specific examples are described as accurately as possible. However, any numerical value inherently must contain a certain amount of error, which is caused by the standard deviation present in the respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all subranges contained therein. For example, a range of "1 to 10" should be considered to include any and all subranges between a minimum value of 1 and a maximum value of 10 (including the endpoint values); that is, all subranges starting with a minimum value of 1 or greater (such as 1 to 6.1) and subranges ending with a maximum value of 10 or less (such as 5.5 to 10).

[0147] It should also be noted that, unless clearly and unequivocally limited to one object, the singular forms used in this specification include the plural forms of their referents. Unless the context indicates otherwise, the term "or" is used interchangeably with the term "and / or".

[0148] As used herein, the term "comprising" or "including" means that various ingredients can be used together in the mixture or composition of the present invention. Therefore, the term "consisting essentially of" or "consisting of" is included in the term "comprising" or "including".

[0149] As used herein, the terms "identity," "percent identity," "homology," or "identical" refer to the 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 in the art (e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444) or by computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI), which are publicly available in the form of BLAST and FASTA. In addition, ENTREZ available at the National Institutes of Health (Bethesda MD) can be used for sequence comparison. When using BLAST or Gapped BLAST programs, the default parameters of each program (e.g., BLASTN, available on the internet website of the National Center for Biotechnology Information) can be used. In one embodiment, a GCG with a gap weight of 1 can be used to determine the percent identity between two sequences. Each amino acid gap is weighted as if it were a single amino acid mispairing between the two sequences. Alternatively, the ALIGN program (version 2.0) can be used, which is a part of the GCG (Accelrys, San Diego, CA) sequence alignment software package.

[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) complementary 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 capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies, diabodies, linear antibodies and multispecific antibodies formed by antibody fragments.

[0151] Traditional antibody structural units typically comprise tetramers. Each tetramer is typically composed of two pairs of identical polypeptide chains, each pair having one "light" chain and one "heavy" chain. Human light chains are classified as kappa and lambda. Heavy chains are classified as μ, δ, γ, α, and ε, and the antibody isotypes are defined as IgM, IgD, IgG, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Thus, as used herein, "isotype" refers to any subclass of an immunoglobulin defined by the chemical and antigenic characteristics of its constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. It should be understood that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses.

[0152] As used herein, "CDR region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., US 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, several, or even all of these regions, which contain the majority of amino acid residues responsible for binding by affinity between an antibody and an antigen or its epitope.

[0153] As used herein, the term "antibody fragment" or "antigen binding fragment" refers to a portion of an antibody analog of a full-length antibody and an antibody that retains the ability to specifically bind to an antigen (e.g., tigit), typically comprising at least a portion of the antigen binding region or variable region of a parent antibody. In some embodiments, the 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). The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed in moles, the antibody fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody to the 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 bond stabilized proteins (dsFv), etc.; linear antibodies, single-chain antibodies (e.g., scFv single antibodies) (technology from Genmab), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (e.g., VH domain antibodies), domain antibodies (technology from Ablynx); multispecific antibodies formed by antibody fragments (e.g., three-chain antibodies, four-chain antibodies, etc.); and engineered antibodies, such as chimeric antibodies (humanized mouse antibodies), heteroconjugate antibodies, etc. These antibody fragments are obtained by conventional techniques known to those skilled in the art, and the utility of these fragments is screened by the same method as for complete antibodies.

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

[0155] In one embodiment, the antibodies of the present invention may be multispecific antibodies, particularly bispecific antibodies, which are sometimes referred to as "diabodies". These are antibodies that bind to two (or more) different antigens or different epitopes on the same antigen. Diabodies can be made in a variety of ways known in the art, for example, by chemical preparation or preparation from hybridomas.

[0156] As used herein, the term "transchromosomal mouse (TC-mAb) TM "Mice)" refers to mice 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 TMThe mice and their offspring can stably maintain human antibody genes and produce human antibodies.

[0157] The antibodies of the present invention are generally isolated or recombinant. When "isolated" is used to describe the various polypeptides disclosed herein, it means that the polypeptide has been identified and separated and / or recovered from the cell or cell culture expressing the polypeptide. Generally speaking, the 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 with different antigenic specificities.

[0158] The present invention further provides variant antibodies. That is, the antibodies of the present invention can be subjected to many modifications, including but not limited to amino acid modifications in the CDR (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 the polypeptide sequence of the parent polypeptide due to at least one amino acid modification. Amino acid modifications can include substitutions, insertions, and deletions. In general, a variant can include any number of modifications, as long as the function of the protein still exists, as described herein.

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

[0160] An epitope can include amino acid residues that are directly involved in binding (also referred to as the immunodominant component of an epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide; in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide. An epitope typically includes at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be validated 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 amino acid polymers of any length of at least two amino acids.

[0162] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to nucleotide polymers of any length of at least two nucleotides, and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and variations thereof.

[0163] As used herein, the terms "pharmaceutical composition," "combination drug," and "drug combination" are used interchangeably and refer to a combination of at least one drug and, optionally, a pharmaceutically acceptable carrier or excipient for achieving a specific purpose. In certain embodiments, a pharmaceutical composition includes a combination separated in time and / or space, as long as they can work together to achieve the purposes of the present disclosure.

[0164] As used herein, "therapeutically effective amount" or "effective amount" refers to a dose sufficient to exert a benefit on the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the person being treated. The prescription of treatment (e.g., dosage determination, etc.) is ultimately the responsibility and decision of general practitioners and other physicians, who generally consider the disease to be treated, the condition of the individual patient, the delivery site, the method of administration, and other factors known to the physician.

[0165] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification sheets and describe an animal, human or non-human being provided for the treatment of the method according to the present invention. The present invention contemplates veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., people under 18 years of age). In addition to humans, patients also include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., domestic pigs, miniature pigs), horses, dogs, cats, cattle and other domestic, farm and zoo animals.

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

[0167] As used herein, the term "pharmaceutically acceptable" means that the compound is physiologically acceptable when administered to humans and does not cause adverse reactions such as gastrointestinal disorders, dizziness or other adverse reactions or systemic adverse reactions similar to these adverse reactions.

[0168] In the present disclosure, "pharmaceutically acceptable carriers" include, but are not limited to, binders (such as microcrystalline cellulose, alginate, gelatin and polyvinyl pyrrolidone), 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), as well as absorption enhancers, flavorings, sweeteners, diluents, coating agents, etc.

[0169] Unless otherwise indicated, the term TIGIT or "T cell immunoreceptor with Ig and ITIM domains" as used herein refers to any native TIGIT from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), and TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIGU, VSTM3, and WUCAM. The term encompasses "full-length", unprocessed TIGIT (e.g., full-length human TIGIT having the amino acid sequence of SEQ ID NO: ) and any form of TIGIT. The term also encompasses naturally occurring TIGIT variants, such as splice variants or allelic variants. The term "TIGIT-related disease" refers to abnormal expression of TIGIT protein or its ligand CD155 in tumors (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)) in subjects (e.g., humans) or in immune-related diseases (e.g., T cell dysfunction diseases). After the anti-TIGIT antibody blocks its binding to the ligand, the anti-TIGIT antibody can inhibit the growth of tumor cells, alleviate the symptoms of other diseases, or cure related diseases, thereby achieving a therapeutic effect on the disease. Such diseases are defined as TIGIT-related diseases.

[0170] "T cell dysfunction disorder" is a T cell disorder or condition characterized by reduced responsiveness to antigenic stimulation. In some embodiments, the T cell dysfunction disorder is characterized by T cell exhaustion. In specific embodiments, the T cell dysfunction disorder is a disorder significantly associated with an inappropriate reduction in signal transduction via OX40 and / or OX40L. In another embodiment, the T cell dysfunction disorder is a disorder in which T cells are unresponsive or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In specific aspects, the reduced responsiveness results in ineffective control of pathogens or tumors expressing immunogens. Examples of T cell dysfunction disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity. In some embodiments, the subject is a human.

[0171] The terms "cancer" and "tumor" are used interchangeably. They refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division may lead to the formation of malignant tumors or the invasion of cells into neighboring tissues, and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0172] "Hematologic 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 most 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 (overlapping with chronic lymphocytic leukemia), Burkitt's lymphoma, mantle cell lymphoma (MCL), mediastinal large B-cell lymphoma, Waldenstmm's macroglobulinemia, nodular marginal zone B-cell lymphoma, splenic marginal zone lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma, and lymphomatoid granuloma. 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. Hematologic malignancies also include leukemias, such as but not limited to secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia and acute lymphocytic leukemia. Hematologic malignancies further include myeloma, such as but not limited to multiple myeloma and smoldering multiple myeloma. Other hematologic and / or B cell or T cell related cancers are encompassed by the term hematologic 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 the GenBank sequence (locus: NM_173799) was synthesized and purchased from Eurofins. The coding region of the extracellular domain ECD corresponding to amino acids (AA) 1-141 of full-length human TIGIT (SEQ ID NO: 2) was amplified by PCR and cloned into an expression vector to produce two recombinant fusion proteins, Trx-huTIGIT-HIS and Gst-huTIGIT-HIS expression plasmids, respectively. To produce recombinant fusion proteins, the recombinant fusion protein (Trx-TIGIT-HIS and Gst-TIGIT-HIS) expression plasmids were transferred into competent Escherichia coli (E. coli gamiB (DE3) pLysS′ Novagen) and cultured. After IPTG induction, the E. coli cells were centrifuged to collect the precipitate. After ultrasonic disruption of the E. coli, the precipitate was obtained by centrifugation. Solubilizing agents were added to dissolve the precipitate, which was then purified by passing through a Ni-NTA column (Qiagen, Ni-NTA Superflow, #30410) and dialyzed. The recombinant protein was checked for purification by PAGE and then stored in small aliquots at -30°C.

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

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

[0183] 2.稳定表达细胞系的建立

[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] (SEQ ID NO: 3).

[0186] 3. Immunization, Hybridoma Fusion and Cloning

[0187] Human TIGIT recombinant protein (Trx-huTIGIT-HIS fusion protein, 100 μg / mouse / primary, 50 μg / mouse / boost) was mixed with Freund's complete adjuvant (FCA, purchased from BD, catalog number 263810, 100 μL / mouse / primary) and used for 6-8 week old transchromosomal mice (TC-mAb) TM mice) and immunized with (Sigma Adjust SAS (purchased from Sigma, catalog number s6322-1vl, 50 μL / mouse / boost) was used for booster immunization injections at intervals of 2-3 weeks. The final immunization did not require an adjuvant, only the 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 node were aseptically removed, and mouse lymphocytes were aseptically separated and extracted. By electrofusion, the lymphocyte population obtained was fused with mouse myeloma cells (1:1, P3X63-Ag8.653, ATCC, #CRL-1580). Fusion cells were placed in the HAT culture medium in 96 well plates, at 37 ℃ and 5% CO 2 , incubated 7 days, replaced with the HT culture medium, and incubated 5 days.

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

[0190] Human TIGIT protein was used to screen supernatants containing specific anti-TIGIT antibodies by ELISA. 96-well plates (Nunc, catalog number 44-2404) were coated with Gst-huTIGIT-HIS fusion protein and Trx-huTIGIT-HIS (50 ng / well), diluted with PBS buffer, and incubated overnight at 4 ° C. The wells were then sealed at room temperature for 0.5 hours using 300 μL of TBS containing skim milk and Tween-20. After washing, 100 μL of supernatant and / or serum were added and incubated at room temperature. In order to detect the specificity of the antibody, horseradish peroxidase-conjugated anti-human IgG antibody (goat anti-human IgG-Fc fragment cross-adsorption antibody HRP-conjugated, BETHYL, #A80-304P) was diluted to the optimal concentration in PBS containing 0.05% Tween-20, and then added at 100 μL / well after washing and incubated at room temperature for 0.5 hours. The plate was washed three times using 300 μL of TBS containing 0.05% Tween-20. 100 μL of a matrix solution containing 0.5 mg / mL OPD and 0.03% H2O2 was added, the plate was incubated at room temperature for 30 minutes, and then 25 μL of 1M H2SO4 (NacalaiTesque, #95626-06) was added and read at 492 nm. Positive well clones were selected and inoculated into new 96-well plates. After 3 days, the supernatant of the new 96-well plates was screened using human TIGIT protein by ELISA. The hybridoma cell line binding to human TIGIT was expanded and cultured for 2-4 days, and 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, secondary detection was performed according to the above method. The secondary positive clones were diluted to the limit, and ICC and flow cytometry were tested again after two weeks, and limiting dilution was performed again.

[0191] Example 2 Sequence Analysis of Anti-TIGIT Antibodies

[0192] After preliminary screening by ELISA, ICC and FACS, the positive hybridoma clones were subcloned by limiting dilution. After verification again, the 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 RNA extracted was rapidly amplified at the 5' cDNA end using a kit (TaKaRa, #Z4858N). The sequence analysis (Eurofins) of the product is shown in Table 1. According to the sequence, a TIGIT antibody expression plasmid was constructed and expressed in HEK293 cells. Eight antibodies were purified by protein A and analyzed by SDS-PAGE, and the antibody purity exceeded 95%. The ten antibodies obtained 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 mAb of the present invention TM Variable region sequences of the VH and VL regions of mouse-derived anti-TIGIT antibodies

[0194]

[0195]

[0196]

[0197]

[0198] CDR predictions for VL and VH were performed using the 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 using different CDR prediction programs, the CDRs of the same VH or the same VL may show slight differences, such as changes in amino acid positions. These different CDRs based on the same VH or VL are also within the scope of the present invention.

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

[0204] The equilibrium dissociation constants (KD) of the eight exemplary antibodies mentioned above for binding to human TIGIT were measured by Fortebio Octet RED96. The measurement method was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013. 5(2): p. 270-8). TM The affinity between mouse-derived anti-TIGIT antibodies and TIGIT-HIS (Biointron, BI120) was measured. An NTA (HIS-tag) sensor was applied. After the sensor was balanced in the assay buffer, human TIGIT-HIS was loaded onto the NTA sensor (fortebio) for affinity measurement. The antigen-loaded sensor was placed in a solution containing the antibody (antibody concentrations were 5, 2.5, 0.83, 0.278, 0.09, 0.03, and 0.01 μg / ml, respectively) until the plateau phase, and then the sensor was transferred to the assay buffer and dissociated for at least 2 minutes for dissociation rate measurement. Dynamic analysis was performed using a 1:1 combination model.

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

[0206] Table 3 Monovalent KD of eight antibodies

[0207]

[0208] The Octet binding test was used to study the epitopes that all human TIGIT antibodies (1B2-8C, 4A042-H3, 4A042-H7, 4B030a, 4B037a, 4B056a, 4A063, 4D035a, 4E061a) bind to human TIGIT, compared to the anti-TIGIT reference antibody Tiragolumab (synthesized according to the Tiragolumab sequence disclosed in the KEGG-DRUG database). The experimental process is as follows: the sensor loaded with TIGIT-HIS (Biointron, BI120) is placed in a solution containing TIGIT antibodies until the plateau phase, and then the sensor is transferred to the analysis buffer to saturation, and then the sensor is transferred to other analytes or buffers containing the reference antibody (Tiragolumab). After binding to the plateau phase, it is eluted. Epitope grouping shows that there is competition between the three candidate clone epitopes compared to Tiragolumab, that is, they combine with the same antigenic epitope of TIGIT. The results are shown in Figure 2A middle.

[0209] Epitope competition exists between the various antibodies of the present invention. 1B2-8C can block the binding of itself to Tiragolumab, 4A042-H3, 4A042-H7, and 4B030a, partially blocks the binding of 4A063, but does not block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; Tiragolumab and 4A042-H3 can block the binding of Tiragolumab, 1B2-8C, 4A042-H7, and 4B030a, but does not block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a. 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; 4A042-H7 can block its own binding to Tiragolumab, 1B2-8C, 4A042-H3, and 4B030a, and partially block the binding of 4B037a and 4A063, but does not block the binding of 4B056a, 4D035a, and 4E061a; 4B030a can block its own binding, partially block the binding of 4B056a and 4A063, and partially block the binding of 4B037a and 4A063. 3, and did not block the binding of 4B037a, 4D035a, and 4E061a; 4B037a and 4A063 could block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a, and partially blocked the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a; 4B056a could block the binding of itself and 4B037a, 4A063, 4D035a and 4E061a, partially blocked the binding of Tiragolumab, 1B2-8C, 4A042-H3 and 4B030a, but did not block the binding of 4A042-H7; 4D035a and 4E061a could block the binding of 4B037a, 4B056a, 4A063, 4D035a and 4E061a, but did not block the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7 and 4B030a. The results are shown in Figure 2B and 2C middle.

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

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

[0212] The cells were washed by adding 200 μL / well wash buffer and centrifuged at 1600 rpm (about 260×g) at 4°C for 3 minutes, and the supernatant was discarded. This was repeated twice. 30 μL of a secondary antibody containing anti-human IgG (Jackson ImmunoResearch, #109-585-190, Alexa Fluor 500, 400 μg) was added to each well. 594AffiniPure goat anti-human IgG, Fcγ fragment specific) wash buffer, cells were incubated at 4°C (on ice) for 1 hour. Washed twice, cells were transferred to a flat-bottom 96-well plate, and analyzed using CytoFLEX S. Antibody concentration was plotted using the logarithm of 10 as the abscissa and the median fluorescence value of the two channels as the ordinate. EC 50 (CHO-huTIGIT and CHO-mkTIGIT) and the peak value of the curve were compared. The results (μg / mL) 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 of the present invention TM Mouse-derived anti-TIGIT monoclonal antibody blocks the binding of TIGIT and CD155

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

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

[0219] IgG1 was used as a negative control antibody to test the blocking ability of eight antibodies on CD155. 50 The results (μg / mL) are shown in Table 5 and Figure 4 middle.

[0220] Table 5 Designated TC-mAbs TM The blocking ability of mouse-derived anti-TIGIT monoclonal antibodies to block the binding of CD155 to TIGIT (IC 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 antibody and anti-PD-1 antibody in vivo, mouse colorectal cancer cell line CT26.WT (5×10 5 cells / mouse) were subcutaneously transplanted into TIGIT humanized BALB / c mice. When the average tumor volume reached 120±50mm 3At 4 hr, mice were randomly divided into groups (n=3) according to tumor volume and administered IgG negative control antibody (anti-HEL human IgG1 isotype, biointron, 200 μg / mouse / time), anti-mPD-1 antibody (InVivoMAb anti-mouse PD-1, lot number: 795720D1, 20 μg / mouse / time), anti-mPD-1 antibody (20 μg / mouse / time) combined with positive reference antibody (Tiragolumab-hIgG1, 200 μg / mouse / time), and anti-mPD-1 antibody (20 μg / mouse / time) combined with 4A063 antibody (4A063-hIgG1, 200 μg / mouse / time) every three days for a total of 6 times. Tumor volume and body weight were measured twice a week.

[0225] The results are shown in Figure 5A and Figure 5B The data are presented in Figure 2, which provides 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. Compared to the positive reference antibody, 4A063 showed significant inhibition of tumor growth (TGI: 44% vs. 92.33%), and there was no significant difference in mean body weight between the groups.

[0226] Example 7 Characterization of the mechanism of action of the in vivo anti-tumor activity of the combination therapy of anti-TIGIT antibody and anti-PD-1 antibody

[0227] In order to study the in vivo mode of action of anti-TIGIT antibodies, after combined treatment with anti-TIGIT antibody 4A063 (hIgG1) and anti-PD-1 antibody, the immune cell infiltration of the tumor was analyzed by flow cytometry. As described in Example 6, mice were inoculated and treated. Three days after six treatments, mice were killed and tumors and spleens were collected. The tumor was dissociated with tumor digestion buffer (1 mg / mL collagenase I and 20 μg / mL DNAase I, sigma), and spleen single cell suspension was obtained directly after spleen grinding. After staining with Fc-block, the cells were stained with anti-CD3 (FITC anti-mouse CD3, Biolegend, 100204) and anti-CD8 (PE anti-mouse CD8a, Biolegend, 100708). After staining with commercial buffer (BD Cytofix / Cytoperm TM After fixation and permeabilization with the ELISA kit (554714), cells were stained with an anti-IFNγ antibody (PerCP / Cyanine 5.5 anti-mouse IFN-γ, Biolegend, 505822). After routine washing and filtration, cells were analyzed by flow cytometry.

[0228] The results are shown in Figure 6A and Figure 6B middle. Figure 6A The results showed that compared with the control group, in vivo treatment of tumors with the anti-TIGIT antibody 4A063 hIgG1 resulted in an increase in the proportion of CD8+ TIL populations in the tumor microenvironment of the combination treatment group (P=0.0335), which was not observed in the single PD-1 antibody group. Figure 6B (The results in the tumor microenvironment are consistent with those in the tumor microenvironment, which also show that the proportion of CD8+TIL populations in the combination treatment group was significantly higher than that in the subtype control group (P=0.0025), and was also higher than that in the single PD-1 antibody treatment group (P=0.0171). This suggests that cytotoxic effector T cells are activated after combination treatment, which may explain the differences between the combination treatment group and the single PD-1 group, as discussed in Example 5. In the comparison within the combination treatment group, compared with the anti-TIGIT reference antibody (Tiragolumab-hIgG1) combination group, the CD8 + The increase in TILs was even more significant. Combining the PD-1 antibody with the anti-TIGIT antibody 4A063 hIgG1 treatment group also improved the function of intratumoral T cells, with an increase in IFNγ production by CD3+CD8+ T cells.

[0229] All publications and patents cited in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the method and composition described in the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, many variations of the described pattern for implementing the present invention are intended to be included in the scope of the claims, and these variations are apparent to those skilled in the relevant art.

Claims

1. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to TIGIT, comprising a heavy chain variable region and a light chain variable region, wherein: (I) the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3 as shown in the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively, and the light chain variable region comprises CDRL1, CDRL2, and CDRL3 as shown in the amino acid sequences of SEQ ID NO: 20, WAS, and SEQ ID NO: 22, respectively; (II) the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3 as shown in the amino acid sequences of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively, and the light chain variable region comprises CDRL1, CDRL2, and CDRL3 as shown in the amino acid sequences of SEQ ID NO:26, WAS, and SEQ ID NO:28, respectively; (III) the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3 as shown in the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, and the light chain variable region comprises CDRL1, CDRL2, and CDRL3 as shown in the amino acid sequences of SEQ ID NO: 32, WAS, and SEQ ID NO: 34, respectively; or, (IV) the heavy chain variable region comprises CDRH1, CDRH2 and CDRH3 as shown in the amino acid sequences of SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, respectively, and the light chain variable region comprises CDRL1, CDRL2 and CDRL3 as shown in the amino acid sequences of SEQ ID NO:38, WAS and SEQ ID NO:40, respectively.

2. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: (I) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; (II) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; (III) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; or (IV) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

10.

3. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The anti-TIGIT antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, which is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; preferably, the heavy chain constant region is the heavy chain constant region of human IgG.

4. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is in a form selected from the group consisting of: F(ab')2, Fab', Fab, Fv, scFv, bispecific antibodies and combinations thereof.

5. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The anti-TIGIT antibody or antigen-binding fragment thereof is coupled to a reagent, wherein the reagent is a detectable label or a cytotoxic agent.

6. A polynucleotide encoding the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. 7 . An expression vector enabling expression of the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 .

8. An engineered cell comprising the polynucleotide according to claim 6 or the expression vector according to claim 7.

9. A pharmaceutical composition comprising the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the polynucleotide according to claim 6, the expression vector according to claim 7 or the engineered cell according to claim 8, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises another immune checkpoint inhibitor; Further preferably, the another immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor and / or a CTLA-4 inhibitor; Further preferably, the another inhibitor is an antibody or an antigen-binding fragment thereof targeting another immune checkpoint.

10. Use of the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the polynucleotide according to claim 6, the expression vector according to claim 7, the engineered cell according to claim 8, or the pharmaceutical composition according to claim 9 for 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 immune disease or an infectious disease, and in particular, the tumor cells are CD155-positive or PVR-positive; and More preferably, the tumor 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.

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