Tigit antibodies and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of Chinese invention patent application No. 2022800819862, entitled "TIGIT antibody and its use".

[0002] Cross-reference to related applications

[0003] This application claims priority to PCT application filed on December 17, 2021, entitled "TIGIT antibody and its use", the disclosure of which is incorporated herein by reference. Technical Field

[0004] This invention relates to antibodies or antigen-binding fragments thereof that specifically bind to TIGIT (a T-cell immune receptor having Ig and ITIM domains), pharmaceutical compositions comprising anti-TIGIT antibodies or antigen-binding fragments thereof, and their uses. Background Technology

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

[0006] TIGIT is a novel 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 on T cells and NK cells, including CD4 cells. + T cells, CD8 +T cells and Treg cells. Under normal conditions, TIGIT expression levels are low, but TIGIT expression increases significantly when T cells and NK cells are activated (J Immunol, 2012, 188:3869-3875, Cancer Cell 26, 923–937, NatImmunol, 19, 723-732). Currently, identified TIGIT ligands include CD155, CD112, and CD113, with CD155 being the major ligand. Crystal structure analysis shows that TIGIT and CD155 form homodimers, which further form heterotetramers through interactions between the ligand and receptor (Proc Natl Acad SciUSA 2012; 109:5399–404). The binding affinity of TIGIT to CD112 or CD113 is significantly lower than that to CD155. CD155 is primarily expressed in dendritic cells, T cells, B cells, macrophages, and non-hematopoietic tissues such as the kidneys, nervous system, and small intestine. Similar to TIGIT, 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 pattern of TIGIT / CD155 is similar to that of the CTLA-4 / CD28 pathway. High-affinity inhibitory receptors and low-affinity activating receptors compete for binding to the same ligand, thereby precisely regulating the immune response. TIGIT bound to CD155 can exert immunosuppressive effects by modulating DC function, suppressing effector T cell activity, interfering with DNAM-1 co-activation, and increasing Treg inhibition (Clinical and Experimental Immunology, 2020 May; 200(2):108-119, Immunity 40, 569–581).

[0007] Several studies in 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 expressed in CD8+ cells. + Highly expressed on T cells, tumor-infiltrating Treg cells, and NK cells. In cancer patients, tumor-infiltrating CD8... +TIGIT expression in T cells and NK cells is generally consistent with high expression of other inhibitory receptors (such as PD-1, LAG-3, and Tim-3) and low expression of DNAM-1. High TIGIT expression is often associated with poor prognosis in malignant tumors. High TIGIT expression in NK cells is associated with disease severity. TIGIT knockout mice show significantly reduced tumor growth and increased survival.

[0008] Due to their macromolecular properties, antibody drugs are often associated with immune-related adverse events (irAEs). In TIGIT knockout mice, there were no spontaneous autoimmune symptoms or hematopoietic developmental disorders. The incidence of autoimmune diseases only increased after crossbreeding with mice predisposed to autoimmune diseases. Animal studies have shown a lower incidence of irAEs during anti-TIGIT mAb administration compared to PD-1 and CTLA-4 mAbs (Oncoimmunology 2018; 7:e1445949). Therefore, antibody drugs targeting TIGIT have a relatively low risk of side effects and are high-quality candidates for clinical anticancer drugs.

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

[0010] After numerous experiments, the inventors of this invention used transchromosomal mice (TC mAb) with all human antibody gene sequences. TM The mouse platform was used to screen and unexpectedly obtained antibodies that specifically bind to TIGIT, which showed excellent affinity for TIGIT and have potential for drug development.

[0011] This invention provides improved medicaments and treatments for cancer and chronic viral infections, the improved medicaments and treatments comprising an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to human TIGIT (huTIGIT). This document provides isolated antibodies, such as monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to huTIGIT and possess desired 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] This invention relates to antibodies that compete with and cross-block the binding of huTIGIT to antibodies having the heavy and light chain variable domain sequences disclosed herein.

[0013] In some embodiments, the anti-TIGIT antibody of the present invention, or its antigen-binding fragment, enhances anti-tumor immune responses, such as antigen-specific T cell responses. In other embodiments, the anti-TIGIT antibody of the present invention, or its antigen-binding fragment, blocks TIGIT-mediated inhibitory signaling, allowing PVR / DNAM co-stimulation of NK cells to increase NK-mediated anti-tumor killing. In another embodiment, the anti-TIGIT antibody of the present invention, or its antigen-binding fragment, depletes a population of regulatory T cells within the tumor that would otherwise suppress the anti-tumor immune response. In another embodiment, the anti-TIGIT antibody of the present invention in the form of IgG1 depletes CD8+ depleted T cells and Tregs, thereby allowing an influx of fresh, non-depleted CD8+ T cells. In some embodiments, the anti-TIGIT antibody of the present invention in the form of IgG1 increases the proportion of CD8+ TIL populations in the tumor microenvironment. In other embodiments, the anti-TIGIT antibody of the present invention, or its antigen-binding fragment, functions through one or more of the mechanisms described above, as these mechanisms are not necessarily mutually exclusive.

[0014] In some embodiments, the anti-TIGIT antibody of the present invention or its antigen-binding fragment does not bind to activating Fcγ receptors (FcγRs), for example in embodiments dependent on enhancing the antitumor activity of TIGIT-expressing cells. In alternative embodiments, the anti-TIGIT antibody of the present invention or its antigen-binding fragment binds to one or more activating FcγRs, for example in embodiments dependent on the 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 an antigen-binding fragment thereof that specifically binds to TIGIT. The anti-TIGIT antibody or antigen-binding fragment includes a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region includes CDRH1, CDRH2, and CDRH3, and the light chain variable region includes CDRL1, CDRL2, and CDRL3.

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

[0017] (a) The CDRH1 comprises a 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 a 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 adding, deleting, or substituting one or more amino acids; and

[0019] (c) The CDRH3 comprises a 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 adding, deleting or substituting one or more amino acids.

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

[0021] (a) The CDRL1 comprises a 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 adding, deleting or substituting one or more amino acids;

[0022] (b) The CDRL2 comprises a 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 adding, deleting, or substituting one or more amino acids; and

[0023] (c) The CDRL3 comprises a 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 invention, wherein

[0025] (a) The CDRH1 comprises sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with sequences 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 sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with sequences 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 sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with sequences 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 sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with sequences 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 sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with sequences 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 sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with sequences 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 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 invention, the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3, and wherein...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0069] (i) CDRH1 contains the sequence of SEQ ID NO:75, CDRH2 contains the sequence of SEQ ID NO:76, and CDRH3 contains the sequence of SEQ ID NO:77; and CDRL1 contains the sequence of SEQ ID NO:72, CDRL2 contains the sequence of SEQ ID NO:73, and CDRL3 contains the sequence of SEQ ID NO:74; or

[0070] (j) CDRH1 contains the sequence of SEQ ID NO:81, CDRH2 contains the sequence of SEQ ID NO:82, and CDRH3 contains the sequence of SEQ ID NO:83; and CDRL1 contains the sequence of SEQ ID NO:78, CDRL2 contains the sequence of SEQ ID NO:79, and CDRL3 contains the sequence of SEQ ID NO:80.

[0071] In some other embodiments of the 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 shown in SEQ ID NO:41, the sequence of CDRH2 is shown in SEQ ID NO:42, and the sequence of CDRH3 is shown in SEQ ID NO:43; and the sequence of CDRL1 is shown in SEQ ID NO:38, the sequence of CDRL2 is shown in SEQ ID NO:39, and the sequence of CDRL3 is shown 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 shown in SEQ ID NO:53, the sequence of CDRH2 is shown in SEQ ID NO:54, and the sequence of CDRH3 is shown in SEQ ID NO:55; and the sequence of CDRL1 is shown in SEQ ID NO:50, the sequence of CDRL2 is shown in SEQ ID NO:51, and the sequence of CDRL3 is shown 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 shown in SEQ ID NO:65, the sequence of CDRH2 is shown in SEQ ID NO:66, and the sequence of CDRH3 is shown in SEQ ID NO:67; and the sequence of CDRL1 is shown in SEQ ID NO:62, the sequence of CDRL2 is shown in SEQ ID NO:63, and the sequence of CDRL3 is shown in SEQ ID NO:64.

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

[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 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 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 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 contains the sequence shown in SEQ ID NO:19, and the light chain variable region contains the sequence shown in SEQ ID NO:18.

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

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

[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 as shown in SEQ ID NO:9, and the sequence of the light chain variable region is as 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 as shown in SEQ ID NO:13, and the sequence of the light chain variable region is as 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 contains the sequence of SEQ ID NO:69, and the light chain variable region contains the sequence of SEQ ID NO:68; or

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

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

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

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

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

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

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

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

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

[0112] (h) The VH includes CDRH1, CDRH2 and CDRH3 of the VH as shown in SEQ ID NO:19, and / or the VL includes CDRL1, CDRL2 and CDRL3 of the VL as 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 includes CDRH1, CDRH2 and CDRH3 of the VH as shown in SEQ ID NO:71, and / or the VL includes CDRL1, CDRL2 and CDRL3 of the VL as shown in SEQ ID NO:70.

[0115] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment of the present invention (a) some antibodies can block the binding of themselves (1B2-8C) to 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... (c) Some antibodies can block the binding of themselves (4A042-H7), 1B2-8C, 4A042-H3 and 4B030a, and 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), and partially block the binding of 4B037a and 4A063, but cannot block the binding of 4B056a, 4D035a and 4E061a. (e) Some antibodies can block the binding of themselves (4B056a, 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, and 4B037a, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a; (g) Some antibodies can block the binding of themselves (4D035a, 4E061a), 4B037a, 4B056a, and 4A063, but not the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a;

[0116] In some embodiments, the anti-huTIGIT antibody of the present invention or its antigen-binding fragment also binds to cynomolgus monkey TIGIT.

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

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

[0119] In some other embodiments of the invention, the anti-TIGIT antibody or its antigen-binding fragment is in the form of the group consisting of: F(ab')2, Fab', Fab, Fv, scFv, bispecific antibodies and combinations thereof.

[0120] The present invention also provides immunoconjugates comprising the anti-TIGIT antibody described herein linked to a reagent (such as a detectable marker or cytotoxic agent).

[0121] In other embodiments, the antigen-binding domain of the antibody of the present invention is present in a bispecific molecule, which further includes an antigen-binding domain that specifically binds to different immunomodulatory receptors, 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 its antigen-binding fragment.

[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 an anti-TIGIT antibody or an antigen-binding fragment thereof (as described in the first aspect), a polynucleotide (as described in the second aspect), a carrier (as described in the third aspect), or a cell and pharmaceutically acceptable carrier (as described in the fourth aspect). The present invention further provides an antibody-drug conjugate comprising the anti-TIGIT antibody or an antigen-binding fragment thereof (as described in the first aspect). Kits containing said anti-TIGIT antibody or an antigen-binding fragment thereof, along with instructions for use, are also provided herein.

[0126] In a sixth aspect, the present invention provides the use of an anti-TIGIT antibody or its antigen-binding fragment (as described in the first aspect), a polynucleotide (as described in the second aspect), a carrier (as described in the third aspect), a cell (as described in the fourth aspect), or a pharmaceutical composition (as described in the fifth aspect) in the manufacture of a medicament for treating TIGIT-related diseases. Preferably, the TIGIT-related disease is a T-cell dysfunction disease; more preferably, the TIGIT-related disease is a tumor, an immune disease, or an infectious disease; even 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 antigen-specific T cell responses, the method comprising contacting T cells with the anti-huTIGIT antibody of the present invention or its antigen-binding fragment thereof, such that the antigen-specific T cell response is enhanced, for example by reducing inhibitory signals that would otherwise attenuate the antitumor response. In some embodiments, the antigen-specific T cells are tumor antigen-specific effector T cells, such as CD8+ T cells, and the increased antitumor activity is caused, for example, by blocking the enhancement of TIGIT-mediated inhibitory effects. The anti-huTIGIT antibody of the present invention or its antigen-binding fragment thereof can also reduce inhibitory signals in NK cells and thus increase their antitumor activity. Not intended to be theoretically limited, the anti-huTIGIT antibody of the present invention increases effector T cell or NK cell function by blocking the binding of TIGIT to PVR, thereby reducing or eliminating inhibitory signals that would otherwise be delivered to the cells. Alternatively or additionally, the anti-TIGIT antibody of the present invention or its antigen-binding fragment thereof can inhibit the interaction between TIGIT and DNAM-1 / CD226, which would otherwise reduce DNAM-1-mediated immune activation.

[0128] This invention provides a method to reduce or deplete T cells in tumors of subjects in need. regs The method comprises administering an effective amount of the anti-huTIGIT antibody of the present invention, wherein the antibody has an effector function or enhanced effector function to reduce T cells in tumors. regs The quantity.

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

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

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

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

[0133] In an eighth aspect, the present invention provides a pharmaceutical composition for treating TIGIT-related diseases, wherein the pharmaceutical composition comprises an anti-TIGIT antibody or an antigen-binding fragment thereof of the first aspect, a polynucleotide of the second aspect, a carrier of the third aspect, or a cell of the fourth aspect. It may be combined with one or more additional therapeutic agents, or used as a bispecific agent together with one or more additional therapeutic agents, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, anti-GITR antibodies, anti-OX40 antibodies, anti-CD73 antibodies, anti-CD40 antibodies, anti-CD137 mAb, anti-CD27 mAb, anti-CSF-1R antibodies and / or anti-CTLA-4 antibodies, TLR agonists, or small molecule antagonists of IDO or TGFβ. In a particular embodiment, anti-TIGIT therapy is combined with anti-PD-1 and / or anti-PD-L1 therapy (e.g., treatment with an antibody that binds to human PD-1 or an antigen-binding fragment thereof, or treatment with an antibody that binds to human PD-L1 or an antigen-binding fragment thereof).

[0134] The present invention also provides a method for detecting the presence of TIGIT in a sample, on cells within the sample (e.g., FACS), or at a specific location (e.g., IHC) in cells or tissues, or a method for sorting cells based on the presence or absence of TIGIT on their surface (e.g., FACS), the method comprising contacting a sample with the anti-TIGIT antibody or its antigen-binding fragment of the present invention, under conditions allowing the formation of a complex between an antibody or its antigen-binding fragment and TIGIT, and detecting the formation of said complex. In some embodiments, the anti-TIGIT antibody for detection is conjugated to a detectable label.

[0135] This invention uses transchromosomal mice (TC-mAb) in which a fully human antibody gene sequence (including gene regulatory sequences) has been transferred. TM A mouse model was used. The target antigen was used to immunize transchromosomal mice to directly obtain fully humanized antibodies. The resulting antibodies do not require subsequent humanization and affinity modification, thereby reducing costs and shortening the development cycle. Furthermore, the antibodies are fully human antibodies derived from transchromosomal mice, which significantly reduces their immunogenicity and is more conducive to drug development. Attached Figure Description

[0136] Figure 1 This demonstrates the use of recombinant huTIGIT protein and the TC-mAb specified in this invention. TM A graph showing the results of 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 this invention is illustrated. TM Results of epitope competition by mouse-derived anti-TIGIT antibodies. See Example 3.

[0138] Figure 3A This illustrates the use of CHO cells expressing human TIGIT and the TC-mAb specified in this invention. TM A graph showing the results of CHO-TIGIT binding assays at different concentrations of mouse-derived anti-TIGIT antibody.

[0139] Figure 3B This demonstrates the use of CHO cells expressing cynomolgus monkey TIGIT (mkTIGIT) and the TC-mAb specified in this invention. TM A graph showing the results of CHO-TIGIT binding assays at different concentrations of mouse-derived anti-TIGIT antibody. See Example 4.

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

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

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

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

[0144] 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 this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All published materials, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0146] Although numerical ranges and parameter approximations are shown within the broad scope of this disclosure, the numerical values ​​shown in specific examples are described as accurately as possible. However, any numerical value must inherently contain a certain amount of error caused by the standard deviation present in the respective measurement. Furthermore, all ranges disclosed herein should be understood to cover any and all subranges contained therein. For example, the range “1 to 10” should be considered, including any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive); that is, all subranges that begin with a minimum value of 1 or greater (e.g., 1 to 6.1) and end with a maximum value of 10 or less (e.g., 5.5 to 10).

[0147] It should also be noted that, unless clearly and explicitly limited to a single object, the singular form as used herein includes the plural form of the object it refers to. Unless the context otherwise indicates, the term "or" is used interchangeably with the term "and / or".

[0148] As used herein, the terms “containing” or “comprising” mean that various ingredients may be used together in the mixtures or compositions of the present invention. Therefore, the terms “consistent with…” or “composed of…” are included in the terms “containing” or “comprising”.

[0149] As used herein, the terms “identity,” “percentage identity,” “homology,” or “identical” refer to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percentage 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. Sequence alignment and comparison 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. MoI. 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 BLAST and FASTA formats. Additionally, ENTREZ, available from the National Institutes of Health (Bethesda MD), can be used for sequence alignment. When using the BLAST or Gapped BLAST program, the default parameters for each program can be used (e.g., BLASTN, available on the website of the National Center for Biotechnology Information). In one implementation, GCG with a vacancy weight of 1 can be used to determine the percentage identity between two sequences. Each amino acid vacancy is weighted as if it were a single amino acid mismatch between the two sequences. Alternatively, the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, CA) sequence alignment software package, can be used.

[0150] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any one of the three CDRs from the immunoglobulin light chain or any one of the three CDRs from the 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, such as bispecific antibodies, single-chain antibodies, biantibodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0151] Traditional antibody structural units typically comprise tetramers. Each tetramer usually consists of two pairs of identical polypeptide chains, each pair having one “light” chain and one “heavy” chain. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, and ε, and 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. Therefore, as used herein, “isotype” means 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 region of the heavy and light chains of an immunoglobulin, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., USDapartment 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 multiple CDRs is used to indicate one or more, or even all, of these regions containing the majority of amino acid residues responsible for binding via 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 a full-length antibody and an antibody analogue that retains the ability to specifically bind to an antigen (e.g., tigit), typically including at least a portion of the antigen-binding region or variable region of the parent antibody. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the 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 fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, FD fragments, complementarity-determining region (CDR) fragments, disulfide bond-stabilized proteins (dsFv), etc.; linear antibodies, single-chain antibodies (e.g., scFv monoclonal antibodies) (from Genmab's technology), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (e.g., VH domain antibodies), domain antibodies (from Ablynx's technology); multispecific antibodies formed from antibody fragments (e.g., triple-chain antibodies, quadruple-chain antibodies, etc.); and engineered modified antibodies, such as chimeric antibodies (humanized mouse antibodies), heteroconjugate antibodies, etc. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the usability of these fragments is screened using the same methods 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., variable domains of the heavy or light chains of mammalian immunoglobulins) that are capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0155] In one embodiment, the antibody of the present invention can be a multispecific antibody, especially a bispecific antibody, which is sometimes also referred to as a "biantibody". These are antibodies that bind to two (or more) different antigens or different epitopes on the same antigen. Biantibodies can be manufactured in a variety of ways known in the art, for example, by chemical methods or from hybridomas.

[0156] As used in this article, the term "transgenic mouse (TC-mAb)" TM "Mouse" refers to a mouse containing an artificial mouse chromosome, wherein the artificial mouse chromosome contains a human antibody heavy chain gene or locus and / or a human antibody κ light chain gene or locus and / or a human antibody λ light chain gene or locus, and at least two mouse endogenous antibody genes or loci corresponding to the human antibody gene or locus are knocked out. TC-mAb TMMice and their offspring can stably maintain human antibody genes and produce human antibodies.

[0157] The antibodies of this invention are typically isolated or recombinant. When “isolated” is used to describe the various polypeptides disclosed herein, it means a polypeptide that has been identified and isolated and / or recovered from cells or cell cultures expressing the polypeptide. Generally, isolated polypeptides will be prepared by at least one purification step. “Isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigen specificities.

[0158] This invention further provides variant antibodies. That is, the antibodies of this invention can be modified in many ways, 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 parent polypeptide sequence due to at least one amino acid modification. Amino acid modifications can include substitution, insertion, and deletion. Generally, variants can include any number of modifications as long as the protein's function remains, as described herein.

[0159] As used herein, the term "epitope" refers to a determinant cluster that interacts with a specific antigen-binding site (called a complementary site) in the variable region of an antibody molecule. Epitopes are groups of molecules (such as amino acid or sugar side chains) and typically possess specific structural and charge characteristics. A single antigen may have more than one epitope.

[0160] Epitopes can comprise amino acid residues that directly participate in binding (also known as the immunodominant component of the epitope) and other amino acid residues that do not directly participate in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide. Epitopes typically comprise at least three, more usually at least five, or eight to ten amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be validated in a simple immunoassay demonstrating the ability of one antibody to block the binding of another antibody to the target antigen.

[0161] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to an amino acid polymer of any length containing at least two amino acids.

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

[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 particular purpose. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided that they can work together to achieve the purposes of this disclosure.

[0164] As used herein, a “therapeutic effective dose” or “effective dose” refers to a dose sufficient to provide benefit to the subject to which it is administered. The actual dose administered, as well as the rate and timing of administration, will depend on the condition and severity of the person being treated. Treatment prescription (e.g., dosage determination, etc.) is ultimately the responsibility and decision of the general practitioner and other physicians, which typically takes into account the disease to be treated, the individual patient’s condition, 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 and describe animals, humans, or non-humans being provided with treatment according to the method of the invention. The invention is contemplated for both veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., domestic pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic, farm, and zoo animals.

[0166] The antibody and chemotherapy preparations of the present invention are administered to subjects according to known methods, such as intravenous administration in the form of a single dose or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local or inhalation routes.

[0167] In this article, the term "pharmaceuticalally acceptable" means that the compound is physiologically acceptable when administered to humans and does not cause adverse reactions such as gastrointestinal symptoms, dizziness or other adverse reactions or systemic adverse reactions similar to these adverse reactions.

[0168] In this disclosure, "pharmaceutically acceptable carriers" include, but are not limited to, binders (such as microcrystalline cellulose, alginate, gelatin, and polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose, and anhydrous lactic acid), disintegrants (such as cross-linked PVP, cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose), lubricants (magnesium stearate, aluminum stearate, talc, polyethylene glycol, and sodium benzoate), wetting agents (such as glycerin), surfactants (such as cetyl alcohol), as well as absorption enhancers, flavoring agents, sweeteners, diluents, coating agents, etc.

[0169] Unless otherwise indicated, the term TIGIT or “T cell immune receptor with Ig and ITIM domains” as used herein refers to any natural TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and protein 9 containing immunoglobulin domains, V-set and protein 3 containing transmembrane domains, VSIGU, VSTM3 and WUCAM. The term encompasses “full-length”, unprocessed TIGIT (e.g., full-length human TIGIT with the amino acid sequence of SEQ ID NO: , as well as any form of TIGIT. The term also encompasses naturally occurring TIGIT variants, such as splice variants or allelic variants. The term “TIGIT-related disease” refers to the aberrant expression of TIGIT protein or its ligand CD155 in tumors (e.g., melanoma, breast cancer, non-small cell lung cancer (NSCLC), colorectal adenocarcinoma (COAD), gastric cancer, acute myeloid leukemia (AML), and multiple myeloma (MM) (Clin Exp Immunol. 2020 May; 200(2):108-119)) or immune-related diseases (e.g., T-cell dysfunction) in a subject (e.g., a human). After blocking its binding to the ligand, the anti-TIGIT antibody can inhibit the growth of tumor cells, or alleviate the symptoms of other diseases, or cure the related diseases, thereby achieving a therapeutic effect. Such diseases are defined as TIGIT-related diseases.

[0170] "T-cell dysfunction syndrome" is a T-cell syndrome or condition characterized by reduced responsiveness to antigen stimulation. In some embodiments, T-cell dysfunction syndrome is characterized by T-cell exhaustion. In a particular embodiment, T-cell dysfunction syndrome is a syndrome markedly associated with an inappropriate reduction in signal transduction via OX40 and / or OX40L. In another embodiment, T-cell dysfunction syndrome is a syndrome in which T cells are unresponsive or have a reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In a particular aspect, reduced responsiveness leads to ineffective control of pathogens or tumors expressing immunogens. Examples of T-cell dysfunction syndrome 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 large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or the invasion of cells into adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0172] "Hematologic malignancies" include lymphoma, leukemia, myeloma, or lymphoma, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include B-cell lymphoma and T-cell lymphoma. 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 lymphoma (overlapping with chronic lymphocytic leukemia), Burkitt lymphoma, mantle cell lymphoma (MCL), mediastinal large B-cell lymphoma, Waldenstmm macroglobulinemia, nodular marginal zone B-cell lymphoma, splenic marginal zone lymphoma, primary exudative 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 lymphoblastic leukemia. Hematologic malignancies further include myelomas, such as, but not limited to, multiple myeloma and smoldering multiple myeloma. Other hematologic and / or B-cell or T-cell-related cancers are covered by the term hematologic malignancies.

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

[0174] In this article, the term "immune-related disease" refers to any immune-related disease in mammals caused, mediated, or otherwise contributed to by components of the mammalian immune system, and also includes diseases that can improve disease progression by stimulating or interfering with the immune response. "Immune-related diseases" include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumors, etc.

[0175] The anti-TIGIT antibody or its antigen-binding fragment of the present invention can be used to treat infections or infectious diseases in subjects (e.g., humans). In some preferred embodiments, the infection or infectious disease is selected from viral infections, bacterial infections, fungal infections, and parasitic infections, including but not limited to HIV, hepatitis viruses, herpesviruses, CMV, EBV, and influenza.

[0176] In the following description, some preferred embodiments and aspects of the present invention will be further described in conjunction with specific examples, and these examples should not be construed as limiting the scope of the invention.

[0177] Example

[0178] Example 1: Generation of anti-TIGIT monoclonal antibody

[0179] 1. Recombinant TIGIT protein for antigen immunization and binding assay

[0180] A cDNA encoding full-length human TIGIT (huTIGIT, SEQ ID NO: 1) based on a GenBank sequence (locus: NM_173799), purchased from Eurofins, was synthesized. The coding region for the extracellular domain ECD corresponding to amino acids (AA)1-141 (SEQ ID NO: 2) of full-length human TIGIT was amplified by PCR and cloned into expression vectors to generate two recombinant fusion protein expression plasmids, Trx-huTIGIT-HIS and Gst-huTIGIT-HIS. To generate the recombinant fusion proteins, the recombinant fusion protein (Trx-TIGIT-HIS and Gst-TIGIT-HIS) expression plasmids were transferred into competent *E. coli* (E. coli gamiB(DE3)pLysS′Novagen) and cultured. After IPTG induction, the *E. coli* cells were centrifuged to collect the precipitate. The precipitate was obtained by centrifugation after sonication of the *E. coli*. Solubilizing agents were added to dissolve the precipitate, and the protein was then purified by passing it through a Ni-NTA column (Qiagen, Ni-NTA Superflow, #30410) and dialyzed. The purification efficiency of the recombinant protein was assessed by PAGE, and the recombinant protein was then stored in aliquots at -30°C.

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

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

[0183] 2. Establishment of a stable expression cell line

[0184] cDNAs encoding full-length human TIGIT (huTIGIT, SEQ ID NO:1) and cynomolgus monkey TIGIT (mkTIGIT, SEQ ID NO:3) were synthesized based on GenBank sequences (NM_73799) and (XM_005548101.2), respectively, and these cDNAs were purchased from Genscript. Following PCR amplification, the DNA products were cloned into the pcDNA3.1 expression vector (Invitrogen) and transduced into the CHO-K1 cell line (JCRB, #JCRB9018) to generate CHO-huTIGIT and CHO-mkTIGIT cell lines. Stable cell lines with high HuTIGIT or mkTIGIT expression were selected by culturing in G418 medium, 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 / booster) was mixed with Freund's complete adjuvant (FCA, purchased from BD, catalog number 263810, 100 μL / mouse / primary) for use in 6-8 week old transchromosomal mice (TC-mAb). TM The mice were given their first immunization and were treated with Sigma Adjust. SAS (purchased from Sigma, catalog number s6322-1vl, 50 μL / mouse / booster) was used for booster immunization, with an interval of 2-3 weeks. The final immunization did not require adjuvants, only Trx-huTIGIT-HIS fusion protein (50 μg / mouse / final). All immunizations were administered via intraperitoneal injection.

[0188] Three days after the final immunization, mice were euthanized, and their spleens and lymph nodes were aseptically removed. Mouse lymphocytes were then aseptically isolated and extracted. The resulting lymphocyte population was fused with mouse myeloma cells (1:1, P3X63-Ag8.653, ATCC, #CRL-1580) via electrofusion. The fused cells were placed in HAT medium in 96-well plates and incubated at 37°C and 5% CO2 for 7 days, followed by incubation in HT medium for 5 days.

[0189] 4. The binding activity of anti-TIGIT antibodies was evaluated 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 for 0.5 h at room temperature with 300 μL of TBS containing skim milk and Tween-20. After washing, 100 μL of supernatant and / or serum was added, and the plates were incubated at room temperature. To determine antibody specificity, horseradish peroxidase-conjugated anti-human IgG antibody (HRP-conjugated goat anti-human IgG-Fc fragment cross-adsorption antibody, BETHYL, #A80-304P) was diluted to the optimal concentration in PBS containing 0.05% Tween-20, added at 100 μL / well after washing, and incubated for 0.5 h at room temperature. Wash the plate three times with 300 μL of TBS containing 0.05% Tween-20. Add 100 μL of matrix solution containing 0.5 mg / mL OPD and 0.03% H2O2, incubate the plate at room temperature for 30 minutes, then add 25 μL of 1M H2SO4 (NacalaiTesque, #95626-06) and read at 492 nm. Select positive clones and seed them into new 96-well plates. After 3 days, screen the supernatant of the new 96-well plates using human TIGIT protein via ELISA. Expand and culture hybridoma cell lines bound to human TIGIT for 2–4 days, and detect ELISA-positive clones using CHO-huTIGIT and CHO-mkTIGIT cells by ICC and flow cytometry, and select positive clones. After several days of culture, perform secondary detection according to the above method. Dilute the secondary positive clones to the limit, and perform ICC and flow cytometry again after two weeks, and perform another limit dilution.

[0191] Example 2: Sequence analysis of anti-TIGIT antibody

[0192] After initial screening by ELISA, ICC, and FACS, positive hybridoma clones were subcloned by limiting dilution. Following re-validation, the cloned hybridoma cells were cultured in 10 cm culture dishes. When the cell density reached 80% to 90%, the cells were collected and resuspended in a solution. RNA was extracted from the suspended cells using a microkit (QIAGEN'#74104). The extracted RNA was rapidly amplified at the 5' cDNA end using a kit (TaKaRa, #Z4858N). Sequence analysis of the products (Eurofins) is shown in Table 1. Based on the sequences, a TIGIT antibody expression plasmid was constructed and expressed in HEK293 cells. Eight antibodies were purified by protein A, and the antibody purity exceeded 95% by SDS-PAGE analysis. The amino acid sequences of the ten obtained antibodies were sequenced, and the heavy chain variable region (VH) and light chain variable region (VL) sequences are shown in Table 1.

[0193] Table 1 TC mAb in this invention TM Variable region sequences of VH and VL regions of mouse-derived anti-TIGIT antibody

[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 for Eight Antibodies

[0200]

[0201]

[0202] It should be noted that when using different CDR prediction procedures, CDRs for the same VH or VL may show slight differences, such as variations in amino acid position. These different CDRs based on the same VH or VL are also within the scope of this invention.

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

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

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

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

[0207]

[0208] The epitopes binding to human TIGIT by all human TIGIT antibodies (1B2-8C, 4A042-H3, 4A042-H7, 4B030a, 4B037a, 4B056a, 4A063, 4D035a, 4E061a) were investigated using the Octet binding assay compared to the anti-TIGIT reference antibody Tiragolumab (synthesized based on the Tiragolumab sequence publicly available in the KEGG-DRUG database). The experimental procedure was as follows: a sensor loaded with TIGIT-HIS (Biointron, BI120) was placed in a solution containing TIGIT antibody until plateau phase. The sensor was then transferred to an analysis buffer until saturated, followed by transfer to other analytes or buffers containing the reference antibody (Tiragolumab). After binding to plateau phase, the sensor was eluted. Epitope grouping revealed competition among the three candidate clone epitopes compared to Tiragolumab, meaning they combined with the same antigenic epitopes of TIGIT. Results are shown in... Figure 2A middle.

[0209] In this invention, epitope competition exists among multiple antibodies. 1B2-8C can block its own binding with Tiragolumab, 4A042-H3, 4A042-H7, and 4B030a, partially block 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 do not block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a. It blocks the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a; 4A042-H7 can block its own binding with Tiragolumab, 1B2-8C, 4A042-H3, and 4B030a, and partially blocks the binding of 4B037a and 4A063, but does not block the binding of 4B056a, 4D035a, and 4E061a; 4B030a can block its own binding, and partially blocks the binding of 4B056a and 4A061a. It binds to 3 and does not block the binding of 4B037a, 4D035a, and 4E061a; 4B037a and 4A063 can block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a, and partially block the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a; 4B056a can block its own binding with 4B037a, 4A063, and 4D035a. Binding to 4E061a partially blocks the binding of Tiragolumab, 1B2-8C, 4A042-H3, and 4B030a, but does not block the binding of 4A042-H7; 4D035a and 4E061a can block the binding of 4B037a, 4B056a, 4A063, 4D035a, and 4E061a, but do not block the binding of Tiragolumab, 1B2-8C, 4A042-H3, 4A042-H7, and 4B030a. Results show that... Figure 2B and 2C middle.

[0210] Example 4 specifies TC-mAb TM The binding activity of mouse-derived anti-TIGIT monoclonal antibody to TIGIT on cell surface

[0211] CHO-huTIGIT cells or CHO-mkTIGIT cells were implanted into 96-well plates. The antibody from Example 2 was diluted to different concentrations and added to the cell-coated 96-well plates (100 μL / well), and incubated at 4°C (on ice) for 1 hour.

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

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

[0214]

[0215]

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

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

[0218] Cells were washed with 200 μL / well of wash buffer and centrifuged at 1600 rpm (approximately 260 × g) at 4 °C for 3 min, discarding the supernatant. This process was 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 to each well, and the cells were incubated at 4 °C (on ice) for 1 h. 30 μL of wash buffer containing streptavidin-594 (fc = 10 μg / mL) was added to each well, and the cells were incubated at 4 °C for 30 min. After one wash, the cells were transferred to flat-bottomed 96-well plates and analyzed using CytoFLEX S. The x-axis was plotted as the logarithm of antibody concentration (μg / mL) to base 10, and the y-axis was plotted as the intermediate fluorescence value corresponding to each antibody concentration. The IC50 values ​​of the curves were analyzed. 50 To differentiate the ability of different antibodies to block the binding between CD155 and TIGIT.

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

[0220] Table 5 specifies the TC-mAb TM The blocking ability (IC50) of mouse-derived anti-TIGIT monoclonal antibody to block the binding of CD155 to TIGIT. 50 )

[0221]

[0222]

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

[0224] To evaluate the synergistic effect of anti-TIGIT antibody and anti-PD-1 antibody in vivo, mouse colorectal cancer cell line CT26.WT (5×10⁻⁶) was used. 5 (1 cell / mouse) was subcutaneously transplanted into TIGIT humanized BALB / c mice. When the average tumor volume reached 120±50 mm... 3Mice were randomly assigned to groups (n=3) based on tumor volume and administered the following antibodies: IgG negative control antibody (anti-HEL human IgG1 isotype, biointron, 200 μg / mouse / dose); anti-mPD-1 antibody (InVivoMAb anti-mouse PD-1, batch number: 795720D1, 20 μg / mouse / dose); anti-mPD-1 antibody combined with positive control antibody (Tiragolumab-hIgG1, 200 μg / mouse / dose) (20 μg / mouse / dose); and anti-mPD-1 antibody combined with 4A063 antibody (4A063-hIgG1, 200 μg / mouse / dose) (20 μg / mouse / dose). These administrations were given every three days for a total of six doses. Tumor volume and body weight were measured twice weekly.

[0225] The results are shown in Figure 5A and Figure 5B The study provided mean tumor volume and mean weight of mice in each group as a function of time. Compared with the positive control antibody, 4A063 showed significant inhibition of tumor growth (TGI: 44% vs. 92.33%), and there was no significant difference in mean weight between groups.

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

[0227] To investigate the in vivo mechanism of action of the anti-TIGIT antibody, tumor immune cell infiltration was analyzed by flow cytometry after combination therapy with anti-TIGIT antibody 4A063 (hIgG1) and anti-PD-1 antibody. Mice were inoculated and treated as described in Example 6. Three days after six treatments, mice were sacrificed and tumors and spleens were collected. Tumors were dissociated with tumor digestion buffer (1 mg / mL collagenase I and 20 μg / mL DNAase I, Sigma), and spleen single-cell suspensions were obtained directly after spleen grinding. After staining with Fc-block, cells were stained with anti-CD3 (FITC anti-mouse CD3, Biolegend, 100204) and anti-CD8 (PE anti-mouse CD8a, Biolegend, 100708). Cells were stained with commercial buffer (BD Cytofix / Cytoperm). TM After fixation and permeabilization using a fixation / permeabilization kit (554714), cells were stained with anti-IFNγ antibody (PerCP / Cyanine 5.5 anti-mouse IFN-γ, Biolegend, 505822). Cells were analyzed by flow cytometry after routine washing and filtration.

[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 anti-TIGIT antibody 4A063 hIgG1 led to an increased proportion of CD8+ TILs in the tumor microenvironment of the combination therapy group (P = 0.0335), which was not observed in the single PD-1 antibody group. Simultaneously, flow cytometry results of T cells in spleen cells (shown in...) Figure 6B Consistent with the results in the tumor microenvironment, this also indicates that the proportion of CD8+ TILs in the combination therapy group was significantly higher than that in the subtype control group (P = 0.0025), and also higher than that in the single PD-1 antibody therapy group (P = 0.0171). This suggests that cytotoxic effector T cells are activated after combination therapy, which may explain the differences between the combination therapy group and the single PD-1 group, as discussed in Example 5. In the comparison within the combination therapy group, compared with the anti-TIGIT reference antibody (Tiragolumab-hIgG1) combination group, the proportion of CD8+ TILs in the 4A063-hIgG1 combination group was significantly higher. + The increase in TILs was more significant. In combination with the anti-TIGIT antibody 4A063 hIgG1 treatment group, the function of intratumoral T cells was also improved, and the production of IFNγ by CD3+CD8+ T cells increased.

[0229] All publications and patents cited in this application are incorporated herein by reference. Various modifications and variations to the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in 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 modes for carrying out the invention are intended to be included within the scope of the claims and will be apparent to those skilled in the art.

Claims

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

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

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

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

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

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

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

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

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

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

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

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

14. Use of the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the polynucleotide according to claim 7, the expression vector according to claim 8, the engineered cell according to claim 9, or the pharmaceutical composition according to any one of claims 10 to 13 for the manufacture of a medicament for the treatment of TIGIT-related diseases; The TIGIT-related diseases mentioned are tumors, among which, The tumor cells are CD155 positive or PVR positive; and The tumors are selected from the group consisting of: melanoma, breast cancer, non-small cell lung cancer, colon adenocarcinoma, gastric cancer, acute myeloid leukemia, and multiple myeloma.

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