Antibodies to tigit

Abstract

To provide antibodies to TIGIT.SOLUTION: The present disclosure provides antibodies that specifically bind to TIGIT. The antibodies have the capacity for substantial activation of T cells and natural killer cells by inhibiting binding of TIGIT to CD155. The antibodies can be used for treatment of cancer and infectious disease, among other applications. Provided herein are, in particular, antibodies that specifically bind to TIGIT, and, among other applications, their use for treatment of cancer and infectious disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Provided herein are, inter alia, antibodies that specifically bind to TIGIT and their uses for the treatment of cancer and infectious diseases, among other uses.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 033,609, filed June 2, 2020, the disclosure of which is incorporated herein by reference in its entirety, including any drawings.

[0003] Incorporating a sequence listing

[0001] This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The attached Sequence Listing text file, named 050658_531001WO_Sequence_Listing_ST25, was created on May 31, 2021, and is 143 KB. [Background technology]

[0004] In tumors, a highly inhibitory microenvironment exists in which T cell and NK cell function is regulated by cell surface checkpoint receptors, allowing cancer cells to evade the immune system. Functional blockade of inhibitory checkpoint receptors, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), has yielded promising results in patients, generating considerable interest in the search for additional co-inhibitory molecules that could act as potential interference targets.

[0005] TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a member of the immunoglobulin superfamily that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail and is a co-inhibitory receptor expressed by regulatory T cells (Tregs), activated T cells, and natural killer (NK) cells. Several groups have reported that TIGIT is a co-inhibitory receptor for CD8 in various tumors. +We reported that TIGIT expression was elevated in tumor-infiltrating lymphocytes (TILs) and Tregs, and also in effector CD8 cells during HIV infection in the blood and SIV infection in lymphoid tissues. + It has also been reported that T cells express higher levels of TIGIT. Furthermore, blocking TIGIT has shown activating activity in human T cell cultures and therapeutic benefits in animal models of different tumors. Therefore, TIGIT may play an important role in antitumor immunity and serve as a promising therapeutic target for the management of cancer and various other diseases and conditions. Therefore, molecules that can interfere with TIGIT binding for beneficial therapeutic purposes are needed. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure relates, inter alia, to anti-TIGIT antibodies.

[0007] Provided herein is an anti-TIGIT antibody or antigen-binding fragment thereof, comprising: (a) a heavy chain (HC) complementarity-determining region (CDR) 1 having at least 80% sequence identity to SEQ ID NO: 36, a HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 37, and a HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 38; and a light chain (LC) CDR1 having at least 80% sequence identity to SEQ ID NO: 39, a light chain (LC) CDR2 having at least 80% sequence identity to SEQ ID NO: 40, and a light chain (LC) CDR3 having at least 80% sequence identity to SEQ ID NO: 41. (b) a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 42, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 43, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 44; and (c) a heavy chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 45, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 46, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 47. (c) a light chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 48, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 49, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 50; and (d) a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 52, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 53, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 54, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 55, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 56;and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 57, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 58, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 59; (e) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 60, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 61, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 64, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 60, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 66, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 69, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 55, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 71, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (h) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 72, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 73, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67;and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65, or (i) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 74, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 75, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65;

[0008] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 1 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 2; (b) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 3 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 4; (c) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 5 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 6. (d) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 7 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 8; (e) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 9 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 10; (f) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 11 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12; (g) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 13 (h) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 15 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 16; (i) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 17 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12; (j) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 76 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 77. (k) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 78 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 77; (l) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 76 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 79; or (m) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 78 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 79;Including.

[0009] Provided herein are anti-TIGIT antibodies or antigen-binding fragments thereof that bind to an epitope comprising at least one of the following amino acid residues of TIGIT: T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope comprising at least one of the following amino acid residues of TIGIT: D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80. In some embodiments, the antibody has the structural features and variable sequences of the CDRs described herein.

[0010] The anti-TIGIT antibody of the present disclosure may be an isolated antibody. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a veneered antibody. In some embodiments, the chimeric antibody comprises a human IgG1 / kappa Fab constant domain. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is a human antibody. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof inhibits binding of TIGIT to CD155.

[0011] Provided herein is a pharmaceutical composition comprising an anti-TIGIT antibody described in this disclosure and a pharmaceutically acceptable carrier.

[0012] Provided herein are methods for treating or effectively preventing cancer, comprising administering to a subject having or at risk of cancer an effective regimen or a therapeutically effective amount of any of the anti-TIGIT antibodies described herein. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is acute myeloid leukemia or adult T-cell leukemia. In some embodiments, the cancer is a solid tumor, non-small cell lung cancer, melanoma, cervical cancer, multiple myeloma, lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, gastric cancer, gastroesophageal junction adenocarcinoma, or esophageal cancer. In some embodiments, the subject also receives tumor-infiltrating T cells. In some embodiments, the subject also receives a vaccine that induces an immune response against the cancer. In some embodiments, the vaccine comprises an antigen or fragment thereof expressed on the surface of cancer cells. In some embodiments, the subject also receives natural killer cells, whose cytotoxicity against the cancer is enhanced by the antibody. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of a cancer cell, whereby the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the anti-TIGIT antibody of the present disclosure. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T cell or a natural killer cell. In some embodiments, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments, the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapy, and surgery. In some embodiments, the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands. In some embodiments, the one or more immune checkpoint receptors or ligands are CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A 2a R,A 2b R.A. 2a / A 2bIn some embodiments, the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, A, arginase, CD39, CD73, IDO, and TDO. 2a R,A 2b R.A. 2a / A 2b R, arginase, CD39, and CD73. In some embodiments, the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimvelelimab, durvalumab, and atezolizumab.

[0013] Provided herein are methods for assisting in the treatment of cancer, comprising administering to a subject with cancer a therapeutically effective amount of any of the anti-TIGIT antibodies described herein. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is acute myeloid leukemia or adult T-cell leukemia. In some embodiments, the cancer is a solid tumor, non-small cell lung cancer, melanoma, cervical cancer, multiple myeloma, lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, gastric cancer, gastroesophageal junction adenocarcinoma, or esophageal cancer. In some embodiments, the subject also receives tumor-infiltrating T cells activated by the antibody. In some embodiments, the subject also receives a vaccine that induces an antibody-enhanced immune response against the cancer. In some embodiments, the vaccine comprises an antigen or fragment thereof expressed on the surface of cancer cells. In some embodiments, the subject also receives natural killer cells whose cytotoxicity against the cancer is enhanced by the anti-TIGIT antibodies of the present disclosure. In some embodiments, the subject is also administered a second antibody that specifically binds to an antigen expressed on the surface of a cancer cell, whereby the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the anti-TIGIT antibody of the present disclosure. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T cell or a natural killer cell. In some embodiments, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments, the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapy, and surgery. In some embodiments, the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands. In some embodiments, the one or more immune checkpoint receptors or ligands are CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A 2a R,A 2b R.A. 2a / A 2bIn some embodiments, the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, A, arginase, CD39, CD73, IDO, and TDO. 2a R,A 2b R.A. 2a / A 2b R, arginase, CD39, and CD73. In some embodiments, the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimvelelimab, durvalumab, and atezolizumab.

[0014] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of the embodiment or aspect. The present invention provides, for example, the following items. (Item 1) An anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to human TIGIT, (a) a heavy chain variable region comprising a heavy chain (HC) complementarity determining region (CDR) 1 having at least 80% sequence identity to SEQ ID NO: 36, a HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 37, and a HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 38; and a light chain variable region comprising a light chain (LC) CDR 1 having at least 80% sequence identity to SEQ ID NO: 39, a LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 40, and a LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 41; (b) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 42, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 43, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 44; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 45, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 46, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 47. (c) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 48, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 49, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 50; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 51, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 52, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 53. (d) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 54, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 55, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 56; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 57, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 58, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 59. (e) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 60, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 61, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 64, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 60, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 66, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65. (g) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 69, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 55, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 71, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (h) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 72, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 73, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; or (i) an anti-TIGIT antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 74, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 75, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65. (Item 2) (a) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 36, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 37, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 38; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising identity to SEQ ID NO: 39, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 40, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 41; (b) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 42, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 43, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 44; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 45, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 46, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 47; (c) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 48, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 49, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 50; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 51, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 52, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 53. (d) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 54, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 55, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 56; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 57, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 58, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 59. (e) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 61, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 64, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 66, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 69, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 55, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 71, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; (h) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 72, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 73, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; or (i) a heavy chain variable region comprising HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 74, HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 75, and HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65. (Item 3) (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 1 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 2; (b) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:3 and a light chain variable region having at least 80% sequence identity to SEQ ID NO:4; (c) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 5 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 6; (d) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 7 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 8; (e) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 9 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 10; (f) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 11, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12; (g) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 13, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 14; (h) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 15, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 16; (i) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 17, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12; (j) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 76, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 77; (k) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 78, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 77; (l) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 76 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 79; or (m) The anti-TIGIT antibody or antigen-binding fragment thereof according to item 1 or 2, comprising a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 78 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 79. (Item 4) 4. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a monoclonal antibody. (Item 5) 5. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric, humanized, or veneered antibody. (Item 6) 6. The anti-TIGIT antibody or antigen-binding fragment thereof according to item 5, wherein the chimeric antibody comprises a human IgG1 / kappa Fab constant domain. (Item 7) 4. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a human antibody. (Item 8) The anti-TIGIT antibody or antigen-binding fragment thereof inhibits the binding of TIGIT to CD155, and optionally, the TIGIT antibody or antigen-binding fragment thereof has an IC50 of about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, measured as in Example 1. 50 8. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody inhibits binding by (Item 9) 9. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 5 or 7 to 8, wherein the antibody further comprises a mutant heavy chain constant region selected from mutant human IgG1, mutant human IgG2, mutant human IgG3, or mutant human IgG4, and optionally a human light chain constant region. (Item 10) 10. The anti-TIGIT antibody or antigen-binding fragment thereof according to item 9, wherein the mutant heavy chain constant region has an enhanced or reduced effector function compared to the wild-type heavy chain constant region. (Item 11) 11. The anti-TIGIT antibody or antigen-binding fragment thereof of item 10, wherein the mutant human IgG heavy chain constant region comprises SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 101. (Item 12) 9. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 5 or 7 to 8, wherein the antibody further comprises a wild-type human IgG heavy chain constant region and, optionally, a human light chain constant region. (Item 13) 13. The anti-TIGIT antibody or antigen-binding fragment thereof of item 12, wherein the wild-type human IgG heavy chain constant region comprises SEQ ID NO: 94. (Item 14) 14. The anti-TIGIT antibody or antigen-binding fragment thereof of item 12 or 13, comprising a human light chain kappa constant region, optionally wherein the human light chain constant region comprises SEQ ID NO: 95. (Item 15) the antibody has a heavy chain and a light chain; (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 92 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 96 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 98 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (d) The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 5 or 7 to 8, wherein the heavy chain has an amino acid sequence comprising SEQ ID NO: 100 and the light chain has an amino acid sequence comprising SEQ ID NO: 93. (Item 16) the antibody or binding fragment thereof (a) Approximately 0.01 × 10 measured by surface plasmon resonance -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 10×10 -11 M, about 1 x 10 -11 M ~ approx. 100×10 -11 M or approximately 1 x 10 -11 M ~ approx. 10×10 -11 having an equilibrium binding constant (KD) of M (b) blocks the binding of soluble human CD155 ligand to cell-surface human TIGIT with a half maximal inhibitory concentration (IC50) of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.6 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured as in Example 1; (c) binds to an epitope comprising at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80; or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80; or (d) The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 15, which is any combination of (a), (b), and (c). (Item 17) 17. The anti-TIGIT antibody or antigen-binding fragment thereof according to Item 16, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of Items 1 to 17 for binding to TIGIT. (Item 18) 18. The anti-TIGIT antibody or antigen-binding fragment thereof of Aspect 16 or 17, wherein the excess antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when measured in a competitive binding assay, and the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93. (Item 19) An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising a heavy chain having an amino acid sequence including SEQ ID NO: 92 and a light chain having an amino acid sequence including SEQ ID NO: 93. (Item 20) A method for inhibiting the binding of TIGIT to CD155, the method comprising contacting TIGIT with the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 19. (Item 21) A method for treating or effectively preventing cancer, comprising administering to a subject having or at risk of cancer an effective regimen or a therapeutically effective amount of any one of the anti-TIGIT antibodies or antigen-binding fragments thereof described in any one of items 1 to 20. (Item 22) 22. The method of claim 21, wherein the cancer is a hematological malignancy, a solid tumor, Merkel cell carcinoma, urothelial cancer, head and neck squamous cell carcinoma, B-cell lymphoma, uterine cancer, cervical cancer, testicular cancer, gastrointestinal cancer, bladder cancer, bone cancer, bone marrow, skin cancer, gallbladder cancer, heart cancer, lung cancer, salivary gland cancer, adrenal cancer, thyroid cancer, ganglionic cancer, cancer of the central nervous system (CNS) and peripheral nervous system (PNS), as well as cancer of the hematopoietic system and cancer of the immune system. (Item 23) 23. The method of item 21 or 22, wherein the subject is administered tumor-infiltrating T cells activated by the antibody or antigen-binding fragment thereof. (Item 24) 24. The method according to any one of items 21 to 23, wherein the subject is administered a vaccine that induces an immune response against the cancer, which is enhanced by the antibody or antigen-binding fragment thereof. (Item 25) 25. The method of claim 24, wherein the vaccine comprises an antigen or a fragment thereof expressed on the surface of a cancer cell. (Item 26) 26. The method according to any one of items 21 to 25, wherein natural killer cells whose cytotoxicity against the cancer is enhanced by the antibody or antigen-binding fragment thereof are administered to the subject. (Item 27) 27. The method of any one of Items 21 to 26, wherein a second antibody against an antigen expressed on the surface of a cancer cell is further administered to the subject, whereby the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the antibody or antigen-binding fragment thereof. (Item 28) 27. The method according to any one of items 21 to 26, wherein the subject is further administered a second antibody against an antigen expressed on the surface of an immune cell. (Item 29) 29. The method of claim 28, wherein the immune cells are T cells or natural killer cells. (Item 30) 29. The method of item 27 or 28, wherein the antigen is CTLA-4, PD-1 or PD-L1. (Item 31) 31. The method according to any one of items 21 to 30, wherein the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapy, and surgery. (Item 32) 32. The method of any one of items 21 to 31, wherein the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands. (Item 33) The one or more immune checkpoint receptors or ligands are CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A 2a R,A 2b R.A. 2a / A 2b 31. The method of item 30, wherein the antibody is selected from the group consisting of R, arginase, CD39, CD73, IDO, and TDO. (Item 34) 33. The method of item 32, wherein the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimvelerimab, durvalumab, and atezolizumab. (Item 35) 20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 19 and a pharmaceutically acceptable carrier. (Item 36) An anti-TIGIT antibody or antigen-binding fragment thereof that binds to an epitope of human TIGIT that includes at least one of the following amino acid residues of SEQ ID NO: 80: T55, Q56, N58, E60, D72, S80, and K82. (Item 37) The antibody or antigen-binding fragment thereof comprises at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80; or (v) The anti-TIGIT antibody or antigen-binding fragment thereof according to Aspect 36, which binds to an epitope including T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80. (Item 38) 38. The anti-TIGIT antibody or antigen-binding fragment thereof according to item 36 or 37, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of items 1 to 19 for binding to TIGIT. (Item 39) 38. The anti-TIGIT antibody or antigen-binding fragment thereof of Aspect 36 or 37, wherein the excess antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when measured in a competitive binding assay, and the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93. (Item 40) 20. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of items 1 to 19, wherein the antibody or antigen-binding fragment thereof binds to an epitope of human TIGIT comprising at least one of the following amino acid residues of SEQ ID NO: 80, i.e., T55, Q56, N58, E60, D72, S80, and K82. [Brief explanation of the drawings]

[0015] [Figure 1A] The amino acid sequences of the mature VH of 21F8 (SEQ ID NO: 1) and the mature VL of 21F8 (SEQ ID NO: 2) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 36), HC-CDR2 (SEQ ID NO: 37), and HC-CDR3 (SEQ ID NO: 38), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 39), LC-CDR2 (SEQ ID NO: 40), and LC-CDR3 (SEQ ID NO: 41), respectively. [Figure 1B]The amino acid sequences of the mature VH of 30M18 (SEQ ID NO: 3) and the mature VL of 30M18 (SEQ ID NO: 4) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 42), HC-CDR2 (SEQ ID NO: 43), and HC-CDR3 (SEQ ID NO: 44), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 45), LC-CDR2 (SEQ ID NO: 46), and LC-CDR3 (SEQ ID NO: 47), respectively. [Figure 1C] The amino acid sequences of the mature VH (SEQ ID NO: 5) and mature VL (SEQ ID NO: 6) of 24F8 are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively. [Figure 1D] The amino acid sequences of the mature VH of 5J24 (SEQ ID NO: 7) and the mature VL of 5J24 (SEQ ID NO: 8) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 54), HC-CDR2 (SEQ ID NO: 55), and HC-CDR3 (SEQ ID NO: 56), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 57), LC-CDR2 (SEQ ID NO: 58), and LC-CDR3 (SEQ ID NO: 59), respectively. [Figure 1E]The amino acid sequences of the mature VH of 21B9 (SEQ ID NO: 9) and the mature VL of 21B9 (SEQ ID NO: 10) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 60), HC-CDR2 (SEQ ID NO: 61), and HC-CDR3 (SEQ ID NO: 62), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 64), and LC-CDR3 (SEQ ID NO: 65), respectively. [Figure 1F] The amino acid sequences of the mature VH of 22B22 (SEQ ID NO: 11) and the mature VL of 22B22 (SEQ ID NO: 12) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 60), HC-CDR2 (SEQ ID NO: 66), and HC-CDR3 (SEQ ID NO: 67), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively. [Figure 1G] The amino acid sequences of the mature VH of 28P24 (SEQ ID NO: 13) and the mature VL of 28P24 (SEQ ID NO: 14) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 69), HC-CDR2 (SEQ ID NO: 55), and HC-CDR3 (SEQ ID NO: 70), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 71), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively. [Figure 1H]The amino acid sequences of the mature VH of 21B16 (SEQ ID NO: 15) and the mature VL of 21B16 (SEQ ID NO: 16) are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 72), HC-CDR2 (SEQ ID NO: 73), and HC-CDR3 (SEQ ID NO: 67), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively. [Figure 1I] The amino acid sequences of the mature VH (SEQ ID NO: 17) and mature VL (SEQ ID NO: 12) of 28O12 are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 74), HC-CDR2 (SEQ ID NO: 75), and HC-CDR3 (SEQ ID NO: 67), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively. [Figure 1J] The amino acid sequences of the mature VH (SEQ ID NO: 76) and mature VL (SEQ ID NO: 77) of Hu24F8.1 are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively. [Figure 1K]The amino acid sequences of the mature VH (SEQ ID NO: 78) and mature VL (SEQ ID NO: 77) of Hu24F8.2 are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively. [Figure 1L] The amino acid sequences of the mature VH (SEQ ID NO: 78) and mature VL (SEQ ID NO: 79) of Hu24F8.3 are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively. [Figure 1M] The amino acid sequences of the mature VH (SEQ ID NO: 76) and mature VL (SEQ ID NO: 79) of Hu24F8.4 are shown. The CDR1, CDR2, and CDR3 amino acid sequences of the VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2, and CDR3 amino acid sequences of the VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively. [Figure 2] This shows that Ch24F8, Ch28O12, and Ch22B22 were able to bind to cyno TIGIT expressed on cynomolgus monkey CD4+ and CD8+ cells. The geometric mean fluorescence intensity (gMFI) was obtained, and the data were expressed as fold gMFI relative to the isotype control. [Figure 3A]1 is a graph showing the binding of humanized anti-TIGIT antibodies to CHO-K1 cells overexpressing human TIGIT. [Figure 3B] 1 is a graph showing the binding of humanized anti-TIGIT antibodies to CHO-K1 cells overexpressing cynomolgus monkey TIGIT. [Figure 4A] 1 is a graph showing binding of humanized anti-TIGIT to CHO-K1 cells overexpressing mouse TIGIT. [Figure 4B] 1 is a graph showing binding of humanized anti-TIGIT to CHO-K1 cells overexpressing rat TIGIT. [Figure 5A] 1 is a graph showing binding of humanized anti-TIGIT antibodies to human non-activated CD8+ T cells. [Figure 5B] 1 is a graph showing binding of humanized anti-TIGIT antibodies to activated human CD8+ T cells. [Figure 6] 1 is a graph showing the inhibition of human CD155 binding to CHO-K1 cells overexpressing human TIGIT by humanized anti-TIGIT antibodies. [Figure 7] 1 is a graph showing the inhibition of human CD155 binding to human TIGIT by humanized anti-TIGIT antibodies in a Jurkat Dual Reporter Cell Line Blockade Assay. [Figure 8] Binding of Fab24F8 to TIGIT is shown. [Figure 9] TIGIT residues that have hydrogen bonds, salt bridges, and van der Waals interactions with Fab24F8 are shown. [Figure 10A] 1 shows that the binding of CD155 to TIGIT is blocked by Fab24F8. 2 shows a schematic diagram of the complex structure of human CD155 (ribbon-shaped) bound to human TIGIT (represented as the molecular surface). [Figure 10B] 10A shows that CD155 binding to TIGIT is blocked by Fab24F8. A superposition of CD155 in the same orientation as in FIG. 10A is shown on a schematic diagram of the crystal structure complex of Fab24F8 (each represented by a molecular surface) bound to TIGIT. [Figure 11] IL-2 response to SEA from one subject in the presence of Hu24F8.2-IgG1, AB122, AB122, and Hu24F8.2-IgG1, or isotype control. Bars and errors represent mean ± standard error of the mean. **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Sidak's multiple comparison test (Hu24F8.2-IgG1 vs. each concentration of IgG1, and AB122 + Hu24F8.2-IgG1 vs. AB122 alone or Hu24F8.2-IgG1 alone). [Figure 12A] Figure 1 shows the IL-2 response of PBMCs from healthy or cancer subjects to SEA in the presence of Hu24F8.2-IgG1 or isotype control. Each symbol represents an individual subject. *p<0.05, paired t-test. [Figure 12B] Figure 1 shows the IL-2 response of healthy subject PBMCs to SEA in the presence of AB122 compared to AB122 and Hu24F8.2-IgG1. Each symbol represents an individual subject. *p<0.05, paired t-test. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure provides, inter alia, antibodies that specifically bind to the extracellular domain of TIGIT. The antibodies of the present disclosure, also referred to herein as "anti-TIGIT antibodies," can inhibit the binding of TIGIT to CD155, thereby activating T cells and / or NK cells. The antibodies can also be used in the treatment of cancer and infectious diseases, among other uses. Additional structural and functional features of the anti-TIGIT antibodies of the present disclosure are described in further detail below.

[0017] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from that understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art using conventional methodology.

[0018] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following alternatives: "A," "B," "A or B," and "A and B."

[0019] The term "antibody" includes intact antibodies and binding fragments thereof, whether they specifically bind to a single antigen or to multiple antigens (e.g., multispecific antibodies such as bispecific antibodies, trispecific antibodies, etc.). Thus, any reference to an antibody should be understood to refer to the intact antibody or a binding fragment, unless the context requires otherwise. Additional functional (e.g., antigen binding) antibodies contemplated in the context of the present disclosure include anti-PD-1, anti-PD-LI, anti-TIM-3, anti-LAG-3, anti-PVRIG, anti-VISTA, anti-CTLA-4, anti-4-1BB, anti-BTLA, anti-CD39, anti-CD73, anti-OX40L, and anti-OX40 fragments.

[0020] The term "binding fragment," which may be used interchangeably with "antigen-binding fragment," as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that specifically binds to an antigen but does not contain the complete native antibody structure. Examples of antigen-binding fragments include diabodies, Fab, Fab', F(ab'), F(ab) c, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), triabodies, tetrabodies, single-chain antibody molecules (scFv), scFv dimers, multispecific antibodies, camelized single-domain antibodies, nanobodies, minibodies, domain antibodies, bivalent domain antibodies, IgNAR, V-NAR, and hcIgG. Typically, binding fragments compete for specific binding with the intact antibody from which they were derived. Binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins.

[0021] "Fab," in reference to an antibody, refers to the portion of an antibody that consists of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0022] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0023] "F(ab')2" refers to a Fab' dimer.

[0024] "Fc," with respect to an antibody, refers to the portion of an antibody consisting of the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is involved in various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0025] "Fv" with respect to an antibody refers to the minimum fragment of an antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0026] A "single-chain Fv antibody" or "scFv" refers to a genetically engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other directly or via a peptide linker sequence (Huston J.S. et al., Proc. Natl. Acad. Sci. USA, 85:5879 (1988)).

[0027] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to a genetically engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0028] "Camelized single domain antibodies," "heavy chain antibodies," or "HCAbs" are antibodies that combine two V H The term "heavy chain antibodies" refers to antibodies containing heavy chains and not containing light chains (Riechmann L. and Muyldermans S., J Immunol Methods. December 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4):277-302 (2001); WO 94 / 04678; WO 94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were derived from camelids (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies have a standard antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428):446-8(1993); Nguyen VK et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. April; 54(1):39-47(2002); Nguyen VK et al. Immunology. May; 109(1):93-101(2003)). The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun. 15(2007)).

[0029] "Nanobody" refers to an antibody fragment consisting of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.

[0030] "Diabodies" comprise small antibody fragments with two antigen-binding sites, the fragments being V in the same polypeptide chain. L Domain-linked V H Domain (V H -V L or V L -V H ) (e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP 404097; WO 93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes).

[0031] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently linked with peptide linkers to create bivalent or multivalent domain antibodies. H The domains can target the same or different antigens.

[0032] In certain embodiments, a "(dsFv)2" is a peptide chain consisting of three peptide chains: one linked by a peptide linker and two V L Two V's bonded to the moiety H Includes parts.

[0033] In certain embodiments, a "bispecific ds diabody" is L1 -V H2 (linked by a peptide linker) to V H1 and V L1 V is linked via a disulfide bridge between H1 -V L2 (also linked by a peptide linker).

[0034] In certain embodiments, a "bispecific dsFv" or dsFv-dsFv'" comprises three peptide chains: heavy chains linked by a peptide linker (e.g., a long flexible linker), each connected via a disulfide bridge, and a V L1 and V L2 V bound to the moiety H1 -V H2 Each disulfide pair of heavy and light chains has a different antigen specificity.

[0035] In certain embodiments, an "scFv dimer" is a dimer of another V H -V L Dimerized V with the moiety H -V L (linked by a peptide linker), and a bivalent diabody or bivalent ScFv (BsFv) comprising one part V H But another part of V L and V to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In another embodiment, an "scFv dimer" is a dimer of V L1 -V H2 V associated with (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker), H1 and V L1 is coordinated, and V H2 and V L2 are coordinated, and each coordinated pair has a different antigen specificity. An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, an isolated antibody is purified to greater than 95% or greater than 99% purity, as determined by methods known in the art.

[0036] As used herein, a "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. A "monoclonal antibody" is not limited to antibodies produced by any particular method. For example, monoclonal antibodies can be produced using hybridoma technology, as well as recombinant, phage display, synthetic, or combinations of such techniques, and other techniques readily known in the art.

[0037] As used herein, the term "humanized antibody" refers to an antibody that contains sequences derived from both human and non-human (e.g., mouse or rat) antibodies.

[0038] As used herein, the term "human antibody" means that the antibody has or consists of amino acid sequences, particularly antigen-binding residues, that correspond to those of antibodies produced by a human or human immune cells, or derived from a non-human source, such as a transgenic non-human animal that utilizes a human antibody repertoire or other human antibody coding sequences. In certain embodiments, a fully human antibody does not contain amino acid residues (particularly antigen-binding residues) derived from a non-human antibody.

[0039] The basic antibody structural unit, as exemplified by native intact antibodies, is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed in association with a cleavable signal peptide. A variable region lacking the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region refers to a light chain variable region lacking the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0040] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. (See generally, Fundamental Immunology (Paul, W., ed., 2nd ed., Raven Press, NY, 1989), Chapter 7) (incorporated by reference in its entirety for all purposes).

[0041] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, a complete, natural antibody has two identical binding sites, a bispecific antibody has two non-identical binding sites, and a trispecific antibody has three non-identical binding sites. The mature variable regions of heavy and light chains all exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both the light chain and the heavy chain contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering system (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number.

[0042] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least three, more usually at least five, or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0043] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay demonstrating the ability of one antibody to compete with the binding of another antibody to a target antigen. An antibody's epitope can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some, but not all, amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0044] Competition between antibodies is determined by an assay in which an antibody inhibits specific binding of a reference antibody to a common antigen under test conditions (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, including values ​​in between) inhibits binding of the reference antibody by at least about 50%, e.g., at least about 75%, 90%, or 99%. In other embodiments, a test antibody competes with a reference antibody if an excess of the test antibody inhibits binding of the reference antibody by at least about any of 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, when measured in a competitive binding assay. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope that is sufficiently close to the epitope bound by the reference antibody to create steric hindrance. The reference antibody can be a commercially available monoclonal antibody with similar functionality to the candidate therapeutic antibody, a polyclonal antibody that functionally interacts with the target protein of interest, or an antibody reconstructed from a sequence available in the public domain. For example, and without limitation, a reference antibody that binds to TIGIT comprises a heavy chain having an amino acid sequence comprising SEQ ID NO:92 and a light chain having an amino acid sequence comprising SEQ ID NO:93.

[0045] As used herein, the terms "specific binding" and "specifically bind" refer to a measurable and reproducible interaction, such as the binding between an antigen (e.g., TIGIT) and an antibody. For example, an antibody that specifically binds to an antigen is one that binds to this target with higher affinity, avidity, more readily, and / or longer duration than it binds to other antigens. The affinity for an antigen and the equilibrium dissociation constant (KD) of the molecule are inversely proportional. A high affinity for an antigen is measured by a low KD value. As used herein, an antibody that specifically binds to an antigen is one that binds to an antigen with a KD value of 10 or more, as measured by surface plasmon resonance. -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or less, or 10 -11 KD for antigens less than or equal to 10 -6 M~10 -13 M or 10 -9 M~10 -13 M or 10 -9 M~10 -12 M or 10 -10 M~10 -13 M or 10 -10 M~10 -12 M or 10 -11 M~10 -13 M or 10 -10 M~10 -11 M or 10 -11 M~10 -12 M. In one embodiment, the term "specific binding" refers to binding of a molecule to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0046] As used herein, "individual" or "subject" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, an "individual" or "subject" is a patient under the care of a physician. Thus, a subject can be a human patient or individual who has, is at risk of, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual who has been diagnosed as being at risk for a condition of interest at or after the time of diagnosis. The term "non-human animal" includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, e.g., sheep, dogs, cows, chickens, etc., and non-mammals, such as amphibians and reptiles.

[0047] As used herein, an amino acid residue in a target amino acid sequence that "corresponds" to or "corresponds to" an amino acid residue in a reference amino acid sequence indicates that the amino acid residue in the target sequence is at a homologous or equivalent position to the listed residue in the reference amino acid sequence. One skilled in the art can determine whether a particular amino acid residue position in a polypeptide, such as a TIGIT polypeptide, corresponds to a homologous reference sequence. For example, the sequence of a TIGIT polypeptide can be aligned with that of a reference sequence using known techniques (e.g., basic local alignment search tool (BLAST), ClustalW2, Structure-based sequences alignment program (STRAP), etc.). Furthermore, the crystal structure coordinates of the reference sequence can be used to aid in determining the three-dimensional structure of homologous polypeptide residues (Stengel et al., Proc. Natl. Acad. Sci. USA, 109:5399-5404, 2012). In another embodiment, equivalent residues can be identified by determining homology at the tertiary structure level. Using such methods, amino acid residues of a TIGIT polypeptide variant can be numbered according to the numbering of corresponding amino acid residue positions in a reference sequence. For example, the amino acid sequence of SEQ ID NO: 80 can be used to determine the numbering of amino acid residue positions for each amino acid residue in a human TIGIT variant or epitope of interest. In some embodiments, one amino acid sequence corresponds to another amino acid sequence if they share at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0048] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions are those that exchange a member of one of these classes for a member of another class.

[0049] Percentage of sequence identity is determined for antibody sequences maximally aligned according to the Kabat numbering convention. After alignment, when a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percentage of sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody regions, divided by the total number of aligned positions in the two regions, not counting gaps, and multiplied by 100 to convert to a percentage.

[0050] A composition or method "comprising" or "including" (or any grammatical variations thereof) one or more listed elements may include other elements not specifically listed. For example, a composition that includes an antibody may include the antibody alone or in combination with other components.

[0051] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" has its original meaning of approximation herein and is used to provide literal support for the exact number it precedes and for numbers that are near or close to the number preceded by the term. When determining whether a number is near or close to a specifically recited number, the near or close unrecited number may be a number that, in the context in which the number is presented, provides a substantial equivalent to the specifically recited number. For example, if the degree of approximation is not clear from the context, "about" means either within ±10% of the provided value, or rounded to the nearest significant figure, and in all cases includes the provided value. When ranges are provided, they include the boundaries.

[0052] As used herein, the term "substantially" and any grammatical variations thereof are broad terms and are used in their ordinary sense, including, but not limited to, almost entirely or largely, but not completely. For example, the term can refer to a numerical value that may not be 100% complete, and the numerical value can be less than 0.1%, less than 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, or less than about 20% of the complete numerical value. For example, a subject antibody or antigen-binding fragment thereof can be substantially derived from a corresponding reference antibody or antigen-binding fragment thereof when the subject antibody or antigen-binding fragment thereof has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the corresponding reference antibody or antigen-binding fragment thereof. In another example, a CDR in a subject antibody can be substantially derived from a corresponding CDR in a reference antibody when the CDR has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the corresponding CDR in the reference antibody. In yet another example, and not limited to this example, a CDR in a subject antibody can be substantially derived from a corresponding CDR in a reference antibody when no more than two amino acids are substituted, deleted, or added in the CDR in the subject antibody compared to the corresponding CDR in the reference antibody.

[0053] It is understood that certain features of the present disclosure that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the present disclosure that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0054] II.Target molecule Unless otherwise indicated, TIGIT refers to human TIGIT (hTIGIT). Cyno TIGIT or cTIGIT refers to cynomolgus monkey TIGIT.

[0055] A representative hTIGIT sequence has been assigned Swiss-Prot accession number Q495A1. The complete hTIGIT sequence has 244 amino acids (SEQ ID NO:80), with amino acids 1-21 being the signal peptide and 22-244 constituting the mature protein (SEQ ID NO:81). Approximately residues 22-141 constitute the extracellular domain of hTIGIT (SEQ ID NO:82). Approximately residues 142-162 constitute the transmembrane domain of hTIGIT, and approximately residues 163-244 constitute the cytoplasmic domain of hTIGIT. In some embodiments, the extracellular domain of hTIGIT is HIS-tagged (SEQ ID NO:83). A representative cynoTIGIT sequence has been assigned Swiss-Prot accession number A0A2K5UW92. The complete cynoTIGIT sequence has 312 amino acids (SEQ ID NO:84). In some embodiments, the extracellular domain of cynoTIGIT is HIS-tagged (SEQ ID NO:85).

[0056] Unless otherwise indicated, CD155 refers to the human form of this protein. A representative human sequence for human CD155 is Swiss-Prot P15151, which is a 417 amino acid protein of which approximately residues 1-20 are a signal peptide, 21-343 constitute the extracellular domain (SEQ ID NO: 86), 344-367 constitute the transmembrane domain, and 368-417 constitute the cytoplasmic domain.

[0057] Unless otherwise clear from the context, reference to one of the above proteins means the entire protein apart from at least the extracellular domain of the protein and usually a cleavable signal peptide.

[0058] III. Antibodies of the Present Disclosure A. Binding Specificity and Functional Properties The present disclosure provides antibodies that specifically bind to TIGIT, more specifically to epitopes within the extracellular domain of the TIGIT protein. In certain embodiments, the anti-TIGIT antibodies of the present disclosure bind to epitopes within the extracellular domain of the TIGIT protein, as measured by surface plasmon resonance (SPR). -8 M or less (e.g., 10 -8 , 10 -9 , 10 -10 In various embodiments, the anti-TIGIT antibodies of the present disclosure have a KD of about 1 x 10 -9 M ~ approx. 1×10 -13 M, or approximately 1 x 10 -9 M ~ approx. 1×10 -12 M, or approximately 1 x 10 -10 M ~ approx. 1×10 -13 M, or approximately 1 x 10 -10 M ~ approx. 1×10 -12 M, or approximately 1 x 10 -11 M ~ approx. 1×10 -13 M, or approximately 1 x 10 -10 M ~ approx. 1×10 -11 M, or approximately 1 x 10 -11 M ~ approx. 1×10 -12They have KDs for TIGIT within the M range. Antibodies designated 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28O12 are nine such representative murine antibodies. Antibodies designated Ch22B22, Ch21B16, Ch28O12, Ch5J24, Ch21B9, Ch24F8, and Ch30M18 are seven such representative chimeric antibodies. Antibodies designated Hu24F8.1, Hu24F8.2, Hu24F8.3, and Hu24F8.4 are representative humanized antibodies. The sequences of the heavy and light chain mature variable regions and CDRs of the murine and humanized antibodies are shown in Tables 1 and 2, respectively. [Table 1] [Table 2]

[0059] Some antibodies of the disclosure bind to the same or overlapping epitope as antibodies designated 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12, or antibodies designated Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Other antibodies with such binding specificity can be generated by immunizing mice with TIGIT or a portion thereof containing the desired epitope and screening the resulting antibodies for binding to the extracellular domain of TIGIT, optionally in competition with 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, 28O12, Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Antibodies can also be screened against mutagenized forms of the TIGIT antigen to identify antibodies that exhibit the same or similar binding profile to mutational variants as 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, 28O12, Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Mutations can be systematically substituted with alanine (or serine, if alanine is already present) one residue at a time, or at more widely spaced intervals, throughout the extracellular domain of the TIGIT antibody, or throughout the section where the epitope is known to reside. In some embodiments, some antibodies of the present disclosure bind to at least one of the following epitope residues of TIGIT: T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO:80. In some embodiments, some antibodies of the present disclosure bind to two, three, four, five, or six of the following epitope residues of TIGIT: T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80. In some embodiments, some antibodies of the present disclosure bind to the following epitope residues of TIGIT: T55, Q56, N58, E60, D72, S80, and K82.In some embodiments, an anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80. In some embodiments, an anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80. In some embodiments, an anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope comprising at least the following amino acid residues of TIGIT: E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80.

[0060] Antibodies having the binding specificity of a selected murine antibody (e.g., 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12) or a selected humanized antibody (e.g., Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4) can also be generated using modifications of the phage display method. See Winter, International Publication No. WO 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of a selected murine antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Strong specific binding to TIGIT (e.g., at least 10 8 , or at least 10 9 M-1 ), and a phage displaying the same heavy chain variable region (i.e., the region identified from the first display library) is selected. The heavy chain variable region from this phage then serves as the starting material for constructing a further phage library, in which each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable region can be obtained, for example, from a library of rearranged human variable light chain regions. Again, a phage displaying strong specific binding to TIGIT is selected. The resulting antibody typically has the same or similar epitope specificity as the murine starting material.

[0061] Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 21F8, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 30M18, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 24F8, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 5J24, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 21B9, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 22B22, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 28P24, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 21B16, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3 derived entirely or substantially from mAb 28O12, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3.The CDRs may be defined by any conventional definition, including Kabat, Chothia, combined Kabat and Chothia, AbM, or Contact definitions, as shown in Table 3 below. [Table 3]

[0062] Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of representative antibodies such as 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12. Also included in the present disclosure are antibodies that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12 in mature heavy and / or light chain variable region, and retain their functional properties, and / or antibodies that differ from each antibody by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Amino acids in the variable region framework that are likely to be important for binding can be identified as described in the humanization section below. Also included are antibodies having at least one, and in some embodiments all six, CDRs as defined by Kabat that are about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding CDRs of 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12.

[0063] In some embodiments, the antibody has one or more of the following characteristics: (i) inhibits binding of human TIGIT to human CD155; (ii) inhibits binding of TIGIT to other ligands such as CD112 and CD113; (iii) enhances antigen-specific T cell responses; (iv) activates natural killer cells; (v) stimulates endogenous T cell activation; and (vi) stimulates the production of one or more immunostimulatory cytokines and / or reduces the production of one or more immunosuppressive cytokines by T cells and other cells of the immune system.

[0064] In some embodiments, the antibodies described herein completely or partially inhibit the binding of TIGIT to CD155. Anti-TIGIT antibodies of the present disclosure can inhibit such interaction with a half maximal inhibitory concentration (IC50) of about 0.1 nM to about 10 nM, or about 0.1 nM to about 8 nM, or about 0.1 nM to about 5 nM, or about 0.1 nM to about 4 nM, or about 0.1 nM to about 3 nM, or about 0.1 nM to about 2 nM, or about 0.1 nM to about 1 nM, as measured in Example 1. In certain embodiments, some anti-TIGIT antibodies of the present disclosure can inhibit the binding of TIGIT to CD155 with an IC50 of about 0.1 nM to about 2 nM, or about 0.2 nM to about 2 nM, as measured in Example 1. In certain embodiments, some anti-TIGIT antibodies of the present disclosure can inhibit binding of TIGIT to CD155 with an IC50 of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured as in Example 1. Some antibodies have a half inhibitory concentration (IC) of approximately 25 to 300 ng / mL, 25 to 75 ng / mL, 25 to 50 ng / mL, 40 to 75 ng / mL, 50 to 75 ng / mL, 50 to 90 ng / mL, 50 to 100 ng / mL, 75 to 100 ng / mL, 50 to 150 ng / mL, 75 to 175 ng / mL, 100 to 200 ng / mL, 125 to 225 ng / mL, 100 to 250 ng / mL, 150 to 300 ng / mL, 175 to 250 ng / mL, 200 to 300 ng / mL, 25 to 275 ng / mL, 250 to 300 ng / mL, 49 + / - 10% ng / mL, 65 + / - 10% ng / mL, or 76 + / - 10% ng / mL, as measured as in Example 1. 50 In other embodiments, the antibody can inhibit such interactions with an IC of at least about 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 125 ng / mL, 150 ng / mL, 175 ng / mL, 200 ng / mL, 225 ng / mL, 250 ng / mL, 275 ng / mL, or 300 ng / mL or more (including concentrations in between). 50and can completely or partially inhibit TIGIT binding to CD155. Furthermore, some antibodies can enhance antigen-specific T cell responses by 1.5 to 3 fold, e.g., about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 fold or more. Alternatively or additionally, some antibodies can increase production of one, two, three, or all of IL-2, IL-6, TNFα, and IFNγ by NK cells and / or T cells by 1.5 to 3 fold, e.g., about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 fold or more. Alternatively or additionally, some antibodies can enhance endogenous T cell activation by 1.5 to 3-fold, e.g., about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3-fold or more. Alternatively or additionally, some antibodies can inhibit cancer or infectious disease, as shown in animal models or clinical trials. Animal models of cancer in which human cancer cells are injected into immunodeficient laboratory animals, such as mice or rats, are widely available.

[0065] In exemplary embodiments, the antibody specifically binds TIGIT and comprises a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3, and a mature light chain region comprising LC-CDR1, LC-CDR2, and LC-CDR3, derived entirely or substantially from antibody 24F8. In various embodiments, the antibody has (i) a denaturation constant of about 0.01 x 10 as measured by surface plasmon resonance; -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 10×10 -11 M, about 1 x 10 -11 M ~ approx. 100×10 -11and / or (ii) is capable of blocking binding of soluble human CD155 ligand to cell-surface human TIGIT with a half maximal inhibitory concentration (IC50) of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured as in Example 1. Alternatively, or in addition to the individual binding and blocking properties described above or a combination thereof, in some embodiments, the antibody binds to at least the following amino acid residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80; or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80.

[0066] Humanization or chimerization of an antibody increases its in vivo half-life relative to the starting murine antibody. The resulting half-life can be, for example, 10 to 50 days in humans. Half-life can be measured by pharmacokinetic studies such as those described by Kim et al., Eur J of Immunol 24:542 (1994).

[0067] B. Non-human antibodies The production of other non-human antibodies against TIGIT, e.g., mice, guinea pigs, primates, rabbits, chickens, or rats, can be achieved, for example, by immunizing animals with TIGIT or a fragment thereof, or cells bearing TIGIT. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such immunogens can be obtained from natural sources, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be fused or otherwise conjugated to a carrier protein and administered. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used, as described below. Complete Freund's adjuvant followed by incomplete adjuvant can be used to immunize laboratory animals. Rabbits or guinea pigs are typically used to generate polyclonal antibodies. Mice are typically used to generate monoclonal antibodies. Antibodies are screened for specific binding to TIGIT. Optionally, the antibody is further screened for binding to a specific region of TIGIT. Such screening can be accomplished by determining the binding of the antibody to a series of deletion mutants of TIGIT and determining which deletion mutants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS, or ELISA.

[0068] C. Humanized Antibodies Reduction or elimination of HAMA (human anti-mouse (also applicable to human anti-rat or human anti-rabbit or human anti-hamster etc.) antibody) responses is a key component in the clinical development of suitable therapeutic agents. See, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described herein, the present disclosure provides antibodies that have been humanized to reduce or eliminate the HAMA response. Variants of these antibodies can further be obtained using conventional methods known in the art, some of which are further described below.

[0069] Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205 and 6,881,557; Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences, a composite of such sequences, a consensus sequence of human antibody sequences, or germline region sequences. Thus, a humanized antibody is an antibody having some or all of the CDRs, completely or substantially, from the donor antibody, and variable region framework sequences and constant region sequences, if present, completely or substantially, from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody heavy chain, and heavy chain variable region framework sequences and heavy chain constant regions, if present, derived substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody light chain, and light chain variable region framework sequences and light chain constant regions, if present, derived substantially from human light chain variable region framework and constant region sequences.Here, as elsewhere in this application, a CDR in a subject antibody is defined as being substantially derived from a corresponding CDR in a reference antibody when at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues (as defined by Kabat) between the respective CDRs are identical, however, at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 100% of the corresponding residues (as defined by Kabat) between the respective CDRs are identical. CDR H2 as defined by Kabat in a subject antibody is substantially derived from the corresponding CDR in a reference antibody when 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. An antibody chain variable region framework sequence or an antibody chain constant region is derived from a substantially human variable region framework sequence or human constant region, respectively, if at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding residues as defined by Kabat are identical.

[0070] Humanized antibodies often incorporate all six CDRs (e.g., as defined by Kabat) from a non-human (e.g., murine) antibody, although they can also be made with fewer than all CDRs (e.g., at least three, four, or five) from the non-human antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0071] In some antibodies, only a portion of the CDRs, i.e., a subset of CDR residues required for binding, called SDRs, are required in the humanized antibody to retain binding. CDR residues that do not contact antigen and are not in the SDRs can be identified from regions of the Kabat CDRs outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987) by molecular modeling and / or empirical analysis, or based on previous studies, as described in Gonzales et al., Mol. Immunol. 41:863, 2004 (e.g., residues H60-H65 in CDR H2 are often unnecessary). In such humanized antibodies, where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid occupying that position can be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to be included reflects a balance of competing considerations. Such substitutions can be advantageous in reducing the number of mouse amino acids in the humanized antibody, thereby reducing potential immunogenicity. However, substitutions can also cause changes in affinity, and significant decreases in affinity can be avoided. The position of substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0072] Although the acceptor may be identical in sequence to the selected human framework sequence, whether derived from a human immunoglobulin or human consensus framework, the present disclosure contemplates that the acceptor sequence may contain pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human consensus framework sequence. These pre-existing substitutions may be minimal, generally only four, three, two, or one amino acid difference compared to the human immunoglobulin sequence or consensus framework sequence.

[0073] The human acceptor antibody sequence can optionally be selected from among many known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between the human acceptor sequence variable region framework and the corresponding variable region framework of the donor antibody chain.

[0074] Specific amino acids from the human variable region framework residues can be selected for substitution based on their possible effects on CDR conformation and / or binding to antigen, exploring such possible effects through modeling, studying the characteristics of amino acids at particular positions, or empirical observation of the effects of substituting or mutagenesing specific amino acids.

[0075] For example, if an amino acid differs between the non-human variable region framework residue and the selected human variable region framework residue, that amino acid may be: (1) It does not bind directly covalently to the antigen; (2) adjacent to the CDR region, (3) If not, when interacting with a CDR region (e.g., within about 6 Å of a CDR region), the human framework amino acid can be replaced by the equivalent framework amino acid from the non-human antibody.

[0076] Other candidates for substitution are acceptor human framework amino acids that are unusual for human immunoglobulins at that position. These amino acids can be substituted with amino acids from the equivalent position of a non-human donor antibody or from the equivalent position of a more typical human immunoglobulin. Other candidates for substitution are acceptor human framework amino acids that are unusual for human immunoglobulins at that position.

[0077] In some embodiments, the humanized anti-TIGIT antibody comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 48 with zero to two amino acid substitutions or deletions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 49 with zero to two amino acid substitutions or deletions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 50 with zero to two amino acid substitutions or deletions; and a framework region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the framework regions of GenBank Accession No. AAV40102.1 or the framework regions of GenBank Accession No. ADX65334.1. and a mature heavy chain variable region comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 51 with zero to two amino acid substitutions or deletions; CDR2 comprising the amino acid sequence of SEQ ID NO: 52 with zero to two amino acid substitutions or deletions; and CDR3 comprising the amino acid sequence of SEQ ID NO: 53 with zero to two amino acid substitutions or deletions; and a mature light chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the framework regions of GenBank Accession No. ACY78416.1 or the framework regions of GenBank Accession No. ADU32611.1. The framework regions of AAV40102.1, ADX65334.1, ACY78416.1, and ADU32611.1 are determined according to the Kabat definition, see Example 2, or see SEQ ID NOs: 76-79, and include the framework regions of AAV40102.1, ADX65334.1, ACY78416.1, and ADU32611.1 and donor CDRs. In some embodiments, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region, and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of full-length antibodies, the mature heavy chain variable region is linked to a heavy chain constant region, and the mature light chain variable region is linked to a light chain constant region.Suitable constant regions are described in further detail in Section III(F). In certain of the above embodiments, the heavy chain constant region has functional FcγR binding ability. In yet further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO:94, and the light chain constant region comprises or consists of SEQ ID NO:95. In certain of the above embodiments, the heavy chain constant region has reduced functional FcγR binding ability. In a further embodiment, the heavy chain constant region comprises or consists of SEQ ID NO:97, and the light chain constant region comprises or consists of SEQ ID NO:95. In still further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO:101, and the light chain constant region comprises or consists of SEQ ID NO:95. In certain of the above embodiments, the heavy chain constant region has enhanced functional FcγR binding ability. In a further embodiment, the heavy chain constant region comprises or consists of SEQ ID NO:99, and the light chain constant region comprises or consists of SEQ ID NO:95.

[0078] In some embodiments, a humanized anti-TIGIT antibody has a mature heavy chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 76, and a mature light chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 77. In some embodiments, any mutations occur at variable region framework residues other than those identified as potentially important for binding. In some embodiments, any mutations are conservative amino acid substitutions. In some embodiments, the antibody comprises a mature heavy chain variable region having SEQ ID NO: 76 and a mature light chain variable region having SEQ ID NO: 77. The Hu24F8.1 antibody of the present disclosure comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 76 and a mature light chain variable region having the sequence of SEQ ID NO: 77. In some of the above embodiments, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region, and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region, and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III(F). In certain of the above embodiments, the heavy chain constant region is capable of inducing Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In still further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions.

[0079] In some embodiments, a humanized anti-TIGIT antibody has a mature heavy chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 78, and a mature light chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 77. In some embodiments, any mutations occur at variable region framework residues other than those identified as potentially important for binding. In some embodiments, any mutations are conservative amino acid substitutions. In some embodiments, the antibody comprises a mature heavy chain variable region having SEQ ID NO: 78 and a mature light chain variable region having SEQ ID NO: 77. The Hu24F8.2 antibody of the present disclosure comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 78 and a mature light chain variable region having the sequence of SEQ ID NO: 77. In some of the above embodiments, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region, and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region, and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III(F). In certain of the above embodiments, the heavy chain constant region is capable of inducing Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In still further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions.In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region induces enhanced Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99 and the light chain constant region comprises or consists of SEQ ID NO: 95.

[0080] In some embodiments, a humanized anti-TIGIT antibody has a mature heavy chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 76, and a mature light chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 79. In some embodiments, any mutations occur at variable region framework residues other than those identified as potentially important for binding. In some embodiments, any mutation is a conservative amino acid substitution. Optionally, the antibody comprises a heavy chain variable region having a sequence comprising SEQ ID NO: 76 and a light chain variable region having a sequence comprising SEQ ID NO: 79. The Hu24F8.3 antibody of the present disclosure comprises a mature heavy chain variable region having a sequence of SEQ ID NO: 78 and a mature light chain variable region having a sequence of SEQ ID NO: 79. In some of the above embodiments, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region, and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region, and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III(F). In certain of the above embodiments, the heavy chain constant region is capable of inducing Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In still further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions.In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region induces enhanced Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99 and the light chain constant region comprises or consists of SEQ ID NO: 95.

[0081] In some embodiments, a humanized anti-TIGIT antibody has a mature heavy chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 78 and a mature light chain variable region having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identity to SEQ ID NO: 79. In some embodiments, any mutations occur at variable region framework residues other than those identified as potentially important for binding. In some embodiments, any mutation is a conservative amino acid substitution. Optionally, the antibody comprises a heavy chain variable region having a sequence comprising SEQ ID NO: 78 and a light chain variable region having a sequence comprising SEQ ID NO: 79. The Hu24F8.4 antibody of the present disclosure comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 76 and a mature light chain variable region having the sequence of SEQ ID NO: 79. In some of the above embodiments, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region, and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region, and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III(F). In certain of the above embodiments, the heavy chain constant region is capable of inducing Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In still further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions.In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region induces enhanced Fcγ receptor (FcγR)-mediated signaling as measured in a commercially available antibody-dependent cell-mediated cytotoxicity reporting bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99 and the light chain constant region comprises or consists of SEQ ID NO: 95.

[0082] In further embodiments of each of the above, the humanized anti-TIGIT antibody has (i) a denaturation rate of about 0.01 x 10 as measured by surface plasmon resonance. -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 10×10 -11 M, about 1 x 10 -11 M ~ approx. 100×10 -11 M, or even about 1 x 10 -11 M ~ approx. 10×10 -11and / or (ii) is capable of blocking binding of soluble human CD155 ligand to cell-surface human TIGIT with a half maximal inhibitory concentration (IC50) of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured as in Example 1. Alternatively, or in addition to the individual binding and blocking properties described above or a combination thereof, in some embodiments, the humanized anti-TIGIT antibody binds to at least the following amino acid residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally T55, Q56, N58, S80, and K82 of SEQ ID NO: 80. (iii) D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80; or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80.

[0083] D. Chimeric and Veneered Antibodies The present disclosure further provides example chimeric and veneered forms of non-human antibodies, particularly the 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28O12 antibodies.

[0084] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., murine) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the non-human antibody and are approximately two-thirds human in sequence.

[0085] A veneered antibody is a type of humanized antibody that retains some, usually all, of the CDRs and some of the nonhuman variable region framework residues of the nonhuman antibody, but replaces other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions in a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially derived from the nonhuman antibody and the variable region framework of the nonhuman antibody is made more human-like by the substitutions. Included in the present disclosure are veneered forms of the 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12 antibodies.

[0086] In some embodiments, the TIGIT chimeric antibody is a mouse-human chimera having a mouse variable domain and a human IgG1 / kappa constant domain. In certain embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 21F8VH (SEQ ID NO: 1) and 21F8VL (SEQ ID NO: 2) domains and a human IgG1 / kappa Fab constant domain (Ch21F8). In certain embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 30M18VH (SEQ ID NO: 3) and 30M18VL (SEQ ID NO: 4) domains and a human IgG1 / kappa Fab constant domain (Ch30M18). In certain embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 24F8VH (SEQ ID NO: 5) and 24F8VL (SEQ ID NO: 6) domains and a human IgG1 / kappa Fab constant domain (Ch24F8). In some embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 5J24VH (SEQ ID NO: 7) and 5J24VL (SEQ ID NO: 8) domains and a human IgG1 / kappa Fab constant domain (Ch5J24). In some embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 21B9VH (SEQ ID NO: 9) and 21B9VL (SEQ ID NO: 10) domains and a human IgG1 / kappa Fab constant domain (Ch21B9). In some embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 22B22VH (SEQ ID NO: 11) and 22B22VL (SEQ ID NO: 12) domains and a human IgG1 / kappa Fab constant domain (Ch22B22). In some embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 28P24 VH (SEQ ID NO: 13) and 28P24 VL (SEQ ID NO: 14) domains and a human IgG1 / kappa Fab constant domain (Ch28P24). In some embodiments, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 21B16 VH (SEQ ID NO: 15) and 21B16 VL (SEQ ID NO: 16) domains and a human IgG1 / kappa Fab constant domain (Ch21B16).In one embodiment, the TIGIT chimeric antibody is a chimeric FabmVH+mVL constructed from mouse 28O12VH (SEQ ID NO: 17) and 28O12VL (SEQ ID NO: 12) domains and a human IgG1 / kappa Fab constant domain (Ch28O12).

[0087] E. Human antibodies Human antibodies against TIGIT can be provided by various techniques, as described below. Some human antibodies are selected by competitive binding experiments, by the Winter phage display method, or by other methods, and have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the Examples. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of TIGIT as the target antigen and / or by screening antibodies against a series of deletion mutants of TIGIT.

[0088] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983), Oestberg, U.S. Pat. No. 4,634,664, and Engleman et al., U.S. Pat. No. 4,634,666, the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., WO 93 / 12227 (1993), U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2030, 2030, 2040, 2041, 2042, 2043, 2044, 2045, 2050, 2051, 2052, 205 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991), and phage display methods (see, e.g., Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, U.S. Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332).

[0089] F. Constant Region Selection The heavy and light chain variable regions of a chimeric, humanized (including veneered), or human antibody can each be linked to at least a portion of a human constant region. In some embodiments, the heavy chain variable domain described in the above section is linked to a portion of a human heavy chain constant region, and the light chain variable domain described in the above section is linked to a portion of a human light chain constant region. In some embodiments, the heavy chain variable domain described in the above section is linked to a portion of a human heavy chain constant region, and the light chain variable domain described in the above section is linked to a full-length human light chain constant region. The heavy chain constant region comprises the Fc (fragment crystallizable) region, which is the tail region of an antibody that interacts with cell surface receptors (Fc receptors) and proteins of the complement system. In some embodiments, the heavy chain variable domain described in the above section is linked to a full-length human heavy chain constant region, and the light chain variable domain described in the above section is linked to a full-length human light chain constant region.

[0090] The choice of constant region (or its cleavage site) depends, in part, on whether effector function is desired or needs to be further enhanced. "Effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding on the C1 complex and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, cell surface receptors (e.g., B cell receptors), and B cell activation. Human antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) according to their heavy chains, each of which serves a different function. IgG consists of four human subclasses (IgG1, IgG2, IgG3, and IgG4), each containing a different heavy chain. These are highly homologous and differ primarily in the hinge region and the degree to which they activate the host immune system. For example, human isotypes IgG1 and IgG3 can mediate complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not or only mediate it at very low levels. The light chain constant region can be of the subclass lambda or kappa. For immunotherapy against cancers or pathogens that do not express TIGIT, in addition to human IgG1 and IgG3, human IgG2 or IgG4, or attenuated forms of human IgG1 with reduced effector function, can be used. In the case of human IgG4, an engineered mutation of S228P (Eu numbering) can be included in the heavy chain to prevent Fab arm exchange. However, for the elimination of TIGIT-expressing cancer cells (e.g., T cell or NK cell tumors) for immunosuppression, human IgG1 or IgG3 can be used. For example, for direct killing of TIGIT-expressing cancer cells (e.g., some hematological malignancies) or for immunosuppression, antibodies with Fc effector function (e.g., human IgG1 or IgG3) can be used. Suitable sequences of human IgG1 or IgG3 are known in the art and include, for example, SEQ ID NO: 94 and the human IgG3 disclosed in US Pat. No. 5,624,821.

[0091] Human constant regions exhibit allotypic and isoallotypic variation among different individuals, i.e., the constant region may differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera that recognize an isoallotype bind to non-polymorphic regions of one or more other isotypes. Reference to a human constant region includes any naturally occurring allotype, or constant regions with any permutation of residues occupying the polymorphic positions of naturally occurring allotypes.

[0092] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in some or all of the molecules. The N-terminal glutamine of the heavy or light chain may be substituted with a glutamic acid residue to prevent the formation of pyroglutamic acid. Substitutions may be made within the constant region to reduce or increase effector functions such as complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA, 103:4005, 2006) or to extend half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position 250 and / or a Leu at position 428 (Eu numbering) to increase the half-life of the antibody.

[0093] In some embodiments, the antibodies described herein comprise a wild-type heavy chain constant region as described above. In some embodiments, the wild-type heavy chain constant region is SEQ ID NO: 94. SEQ ID NO: 92 is a representative heavy chain amino acid sequence comprising the wild-type constant region of SEQ ID NO: 94. In other embodiments, the antibodies described herein have a variant of the wild-type heavy chain constant region (or a truncation thereof) selected from a mutant human IgG1, a mutant human IgG2, a mutant human IgG3, or a mutant human IgG4. In some embodiments, the mutant heavy chain constant region is SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 101. SEQ ID NOs: 96, 98, and 100 are representative heavy chain amino acid sequences comprising a mutant heavy chain constant region.

[0094] Some antibodies of the present disclosure are engineered by introducing constant region mutations to reduce effector functions such as CDC and ADCC or antibody-dependent cellular phagocytosis (ADCP) compared to the same antibody without the mutations. In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% compared to the antibody without the mutations. Other assays are described by Shields et al., 2001 J. Biol. Chem., Vol. 276, pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.

[0095] Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., U.S. Patent No. 6,624,821). In some embodiments, alanine residues are used for substitutions, such as the L234A / L235A double mutation, to reduce effector function. Other combinations of mutations associated with reduced effector function include L234A / L235A / G237A, E233P / L234V / L235A / ΔG236, A327G / A330S / P331S, K322A, L234A, and L235A, L234F / L235E / P331S (Eu numbering). Optionally, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, e.g., U.S. Patent No. 5,624,821). Two amino acid substitutions in the complement C1q binding site at Eu index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of IgG2 residues at positions 233-236 with human IgG1 and IgG4 residues at positions 327, 330, and 331 reduces ADCC and CDC (see, e.g., Armour KL et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL et al., 2001 J Biol Chem. 276(9):6591-604). N297A, N297Q, or N297G (Eu numbering) mutations reduce glycosylation and thereby reduce effector function.

[0096] In some embodiments, antibodies of the present disclosure can be engineered to enhance Fc effector function. For example, FcγR binding can be enhanced by amino acid engineering. In some embodiments, this can be achieved by substituting one or more amino acids in the Fc region. Desired mutations can be determined, for example, by alanine scanning or rational design and library screening. IgG variants with enhanced FcR binding and enhanced effector function can be identified using these approaches. Alternatively, several mutations to the Fc receptor region are known in the art and are described, for example, in Smith P. et al. (2012) PNAS 6181-6186.

[0097] In some embodiments, the antibodies described herein comprise a modified IgG1 constant domain that increases the ability of the antibody to mediate ADCC compared to a wild-type IgG1 that does not comprise the modification. The modified IgG1 domain can be characterized by amino acid substitutions at one or more of L235V, S239D, F243L, R292P, A330L, I332E, P396L (Eu numbering). In other embodiments, the modified IgG1 domain is characterized by substitutions at S239D, A330L, and I332E (Eu numbering). In some embodiments, a therapeutically effective amount of an antibody described herein can induce cell death in at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of TIGIT-expressing cells within 1, 2, or 3 hours, as assessed by methods described in the art.

[0098] Alternatively, glycoform perturbation can be used to enhance Fc-mediated therapeutic antibody function. N-linked Fc glycosylation on IgG1 antibodies is important for effector function. Sialylation, galactosylation, bisecting sugars, and fucosylation can all affect the binding and activity of IgG molecules. Controlling the glycosylation pattern on therapeutic antibodies can be achieved in many different ways. The type of cells producing the recombinant antibody and their culture conditions can affect the glycosylation and activity of the therapeutic antibody. Furthermore, bioreactor conditions and downstream processing can also affect glycan microheterogeneity. Hypofucosylated or afucosylated antibodies have been shown to enhance Fc-mediated properties. Numerous methods for achieving this reduction in fucose levels through glycoengineering are well known in the art. One method is to manipulate enzymes involved in post-translational modification of antibodies. This may involve overexpression of glucosidases such as β-1-4-N-acetylglucosaminyltransferase III, knockout of fucosyltransferases, or the use of naturally fucose-deficient cell lines or cell lines mutated to express reduced fucosylation levels. Additionally, inhibitors of N-linked glucosidases such as castanospermine can be used to obtain IgG molecules with reduced fucose.

[0099] In some embodiments, amino acid engineered variants can have broader enhanced affinity for multiple FcγRs, while glycoform engineered antibodies can generally have more specific affinity for enhanced FcγRIIIa binding. Glycoforms interact with proximal amino acids on the Fc portion, and substitution of amino acids that contact Ig oligosaccharides can result in different glycoform structures.

[0100] G. Expression of Recombinant Antibodies Chimeric, humanized (including veneered), and human antibodies are typically produced by recombinant expression. Accordingly, the present disclosure also provides polynucleotides encoding the anti-TIGIT antibodies of this Section IIIA-G, vectors containing the polynucleotides, and host cells containing the vectors.

[0101] Polynucleotides encoding the anti-TIGIT antibodies of the present disclosure can be inserted into vectors for amplification, expression, or further optimization. Many vectors are available. In some embodiments, vector systems include mammalian, bacterial, and yeast systems, including, but not limited to, plasmids such as pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, and pACT2.2, as well as other experimentally available and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. For expression, recombinant polynucleotide constructs typically contain expression control sequences operably linked to the coding sequence of the antibody chain, including naturally associated or heterologous promoter regions. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence, and the recovery and purification of the recombinant antibody.

[0102] Vectors containing polynucleotide sequences encoding the anti-TIGIT antibodies of the present disclosure can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the polynucleotide sequences in the vectors herein include prokaryotic and eukaryotic cells. Non-limiting examples of suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive bacteria, for example, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, e.g., P. aeruginosa, and Streptomyces. In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding anti-TIGIT antibodies. Non-limiting examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, etc., and Aspergillus hosts such as A. nidulans and A. niger.Suitable host cells can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts. In some embodiments, mammalian cells are used as host cells for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, "From Genes to Clones" (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2 / 0 and NS0. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. In some embodiments, the expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0103] Host cells are transformed with the above-described expression or cloning vectors to produce anti-TIGIT antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Upon expression, antibodies can be purified according to standard procedures in the art, including HPLC purification, column chromatography, and gel electrophoresis (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0104] IV. Therapeutic uses The anti-TIGIT antibodies of the present disclosure can be used to enhance immune responses in the treatment of cancer and infectious diseases. Diseases that can be treated by the antibodies of the present disclosure include, but are not limited to, hematological malignancies, solid tumors, Merkel cell carcinoma, urothelial cancer, head and neck squamous cell carcinoma, B-cell lymphoma, uterine cancer, cervical cancer, testicular cancer, gastrointestinal cancer (e.g., esophageal cancer, gastroesophageal junction cancer, oropharyngeal cancer, stomach cancer, small intestine or large intestine cancer, colon cancer, or rectal cancer), bladder cancer, bone marrow cancer, skin cancer, gallbladder cancer, heart cancer, lung cancer, salivary gland cancer, adrenal cancer, thyroid cancer, central nervous system (CNS) cancer, and peripheral nervous system (PNS) cancer, as well as cancers of the hematopoietic and immune systems (e.g., spleen or thymus).

[0105] The present disclosure also provides methods for treating or preventing other cancer-related diseases, disorders, or conditions, including, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers (e.g., epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, and papillomavirus), adenocarcinoma, teratocarcinoma, chemically-induced cancer, metastasis, and angiogenesis. In certain embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, or leukemia. The use of the term cancer-related diseases, disorders, and conditions is meant to refer broadly to conditions directly or indirectly associated with cancer, including precancerous conditions such as angiogenesis and dysplasia. In certain embodiments, the cancer may be metastatic or at risk of becoming metastatic, or may occur in spread tissues, including cancers of the blood or bone marrow (e.g., leukemia).

[0106] Such cancers may or may not express TIGIT or CD155. Antibodies against TIGIT are effective against cancers that do not express TIGIT, because inhibiting the interaction of TIGIT with CD155 stimulates an immune response against such cancers. Examples of hematological malignancies include leukemia, lymphoma, and myeloma, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin's and non-Hodgkin's lymphoma, and multiple myeloma. Examples of solid tumors include, but are not limited to, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, and brain tumors (e.g., gliomas such as glioblastoma).

[0107] The methods of the present disclosure can be carried out in an adjuvant setting. An "adjuvant setting" refers to a clinical setting in which a subject has a history of a proliferative disease, particularly cancer, and has generally (but not necessarily) responded to treatment, including, but not limited to, surgery, radiation therapy, and / or chemotherapy. However, due to the history of the proliferative disease, these subjects are considered to be at risk for developing the disease. Treatment or administration in an "adjuvant setting" refers to a subsequent form of treatment. In some embodiments, provided herein are methods of treating or effectively preventing cancer, comprising administering a therapeutically effective amount of any of the antibodies disclosed herein in an adjuvant setting to a subject having or at risk of cancer.

[0108] The methods provided herein can also be performed in a "neoadjuvant setting," i.e., the methods can be performed prior to primary / definitive treatment. In some aspects, the subject has been previously treated. In other aspects, the subject has not been previously treated. In some aspects, the treatment is a first-line treatment. In some embodiments, provided herein are methods of treating or effectively preventing cancer, comprising administering to a subject having or at risk of cancer a therapeutically effective amount of any of the antibodies disclosed herein in a neoadjuvant setting.

[0109] Other diseases treatable by the antibodies of the present disclosure include viruses, bacteria, fungi, protozoa, and other pathogens (e.g., hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, and the like). Infectious diseases include those caused by: HIV, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, HIV, SIV, and arboviral encephalitis viruses, chlamydia, rickettsia, mycobacteria, staphylococci, treptocci, pneumococci, meningococci and conococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospirosis, and Lyme burgdorferi.

[0110] A. Antibody Administration The antibodies described herein are administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further progression, and / or improves at least one sign or symptom of the disease. If the subject already suffers from a disorder, the regimen can be referred to as a therapeutically effective regimen. If the subject is at increased risk for the disease compared to the general population but has not yet experienced symptoms, the regimen can be referred to as a prophylactically effective regimen. In some instances, therapeutic or prophylactic efficacy can be observed in an individual subject compared to historical controls or past experience in the same subject. In other instances, therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials in a population of treated subjects compared to a control population of untreated subjects.

[0111] In some cases, the subject is identified as PD-L1 positive, CD155 positive, TIGIT positive, high frequency of MSI, having infiltrating T cells, having activated T cells, having high levels of molecules associated with antigen processing and presentation, high TMB, or any combination thereof. In some embodiments, patients are selected for treatment with the antibodies described herein based on, for example, high expression of TIGIT on CD8+ cells and / or CD4+ cells and / or NK cells compared to a control population. In some cases, the subject is identified as having a cancer caused by an oncogene, such as TP53, VHL, KRAS, BRAF, MET, FUBP1, RAC1, EGFR, CDK4, CTCF, PGR, RET, RASA1, JAK1, PHF6, NF1, CIC, ARID1A, ZFHX3, ZCCHC12, GNA11, SMAD4, USP9X, CDKN2A, FAT1, PIK3R1, SCAF4, PMS2, RNF43, SMC1A, BCOR, FGFR2, COL5A1, ATM, KMT2B, CTNNB1, MYC, RAD21, PTEN, AXL, HIF1A, EPAS1, PAK4, RHOB, TBL1XR1 , KEAP1, ZFP36L2, FGFR3, FOXA1, FLT3, TRAF3, RNF111, PPP2R1A, TXNIP, STAG2, RIT1, TGIF1, FOXQ1, ATR, CYSLTR2, PCBP1, PIK3R2, ASXL1, HIST1H1C, KLF5, PIK3CB, SPOP, MECOM, CACNA1A, CTNND1, DACH1, XPO1, ZNF750, FBXW7, MUC6, KDM6A, GATA3, ZBTB20, PIK3CA, RB1, SOX17, SMARCA4, KIT, CHD8, CHD4, and APOB.

[0112] In some aspects, any of the methods described herein comprises administering a therapeutically effective amount of one or more of the anti-TIGIT antibodies described herein to a subject in need thereof. As used herein, a "therapeutically effective amount" or "therapeutically effective dose" of an anti-cancer therapy (such as any of the anti-TIGIT antibodies described herein) is an amount sufficient to produce a beneficial or desired result. In therapeutic use, beneficial or desired results include, but are not limited to, a reduction in one or more symptoms of cancer, an improvement in the quality of life of a subject afflicted with cancer, a reduction in the dose of other drugs required to treat cancer, an enhancement of the effect of another drug, such as by targeting, a delay in disease progression, and / or an increase in survival. An effective dose may be administered in one or more administrations. For purposes of this disclosure, an effective dose of an anti-cancer therapy is an amount sufficient to achieve therapeutic or prophylactic treatment, either directly or indirectly. As understood in the clinical context, a therapeutically effective dose of an anti-cancer therapy may or may not be achieved in conjunction with another anti-cancer therapy.

[0113] A typical dosage of any of the antibodies described herein is about 0.1-20 mg / kg or 0.5-5 mg / kg body weight (e.g., about 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg), or 10-1600 mg as a fixed dose. g (e.g., less than or equal to 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, or 1600 mg, inclusive of any value therebetween). In one embodiment, the antibodies described herein are administered in an amount of about 300-1500 mg every three weeks. In another embodiment, the antibodies described herein are administered in an amount of about 300-1800 mg every four weeks. The dosage will depend, among other factors, on the subject's condition and response to previous treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0114] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intratumoral, topical, intranasal, or intramuscular. In some embodiments, administration to the systemic circulation is by intravenous or subcutaneous administration. Intravenous administration can be by infusion over a period of time, such as 30 to 90 minutes.

[0115] The frequency of administration depends, among other things, on the half-life of the antibody in circulation, the condition of the subject, and the route of administration. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals, depending on changes in the subject's condition or the progression of the disorder being treated. In one embodiment, the frequency can be a two-week cycle. In another embodiment, the frequency can be a three-week cycle. In another embodiment, the frequency is a four-week cycle. In another embodiment, the frequency is a six-week cycle. An exemplary frequency for intravenous administration is between weekly and quarterly, depending on the sequential cause of treatment, although more or less frequent administration is also possible. For subcutaneous administration, an exemplary frequency for administration is daily to monthly, although more or less frequent administration is also possible.

[0116] The dosage depends on whether the disorder is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often sufficient. In some cases, a single bolus dose, optionally in divided form, is sufficient for acute disorders or acute exacerbations of chronic disorders. Treatment can be repeated for recurrence or acute exacerbations of acute disorders. For chronic disorders, the antibody can be administered at regular intervals, for example, weekly, biweekly, quarterly, or every six months, for at least 1, 5, or 10 years, or for the life of the subject.

[0117] Treatments comprising anti-TIGIT antibodies improve median progression-free survival or overall survival of subjects with cancer by at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 1 0%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%, or extending any of these periods by 2 weeks, 1, 2 or 3 months, or 4 or 6 months, or even 9 months or 1 year can alleviate the disease. Additionally or alternatively, treatment comprising an anti-TIGIT antibody may increase a subject's complete response rate, partial response rate, or objective response rate (complete + partial) by at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 13 %, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% increase. Control subjects receive the same treatment as the subjects receiving the anti-TIGIT antibody, except for the anti-TIGIT antibody. Thus, control subjects can receive a placebo alone or a combination of a placebo and some other chemotherapeutic agent other than the anti-TIGIT antibody, if the subject receiving the anti-TIGIT antibody is also receiving that chemotherapeutic agent.

[0118] The anti-TIGIT antibodies disclosed herein can enhance NK cell-mediated cytotoxicity of CD155-expressing cells (e.g., without limitation, K562 cells) by any of about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, or more relative to the amount of NK cell-mediated cytotoxicity of CD155-expressing cells in the absence of one of the anti-TIGIT antibodies disclosed herein.

[0119] Typically, in a clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial), the increase in progression-free survival and / or response rate of subjects treated with an anti-TIGIT antibody compared to control subjects is statistically significant, for example, at the p=0.05 or 0.01 or even 0.001 level. Complete response rates and partial response rates are determined by objective criteria commonly used in cancer clinical trials, for example, as listed by the National Cancer Institute and / or the Food and Drug Administration, and may include, among other things, measures of tumor volume, number of tumors, metastasis, survival time, and quality of life.

[0120] Pharmaceutical compositions for parenteral administration are sterile, substantially isotonic, and may be manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation will depend on the route of administration selected. For injection, antibodies may be formulated in aqueous solutions, such as physiologically compatible buffers such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). Solutions may contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., pyrogen-free distilled water, before use. The concentration of the antibody in a liquid formulation may vary, e.g., from about 10 to 150 mg / mL. In some formulations, the concentration is from about 20 to 80 mg / mL.

[0121] B. Combination Therapy The present disclosure contemplates the use of anti-TIGIT antibodies, alone or in combination with one or more active therapeutic agents. The additional active therapeutic agent may be a small chemical molecule; a macromolecule such as a protein, antibody, peptibody, peptide, DNA, RNA, or a fragment of such a macromolecule; or a cell therapy or gene therapy. Combination therapy may target different but complementary mechanisms of action, thereby having a synergistic therapeutic or preventative effect against the underlying disease, disorder, or condition. Additionally, or alternatively, combination therapy may allow for a reduced dose of one or more of the agents, thereby ameliorating, reducing, or eliminating adverse effects associated with one or more of the agents.

[0122] The active therapeutic agents in such combination therapy can be formulated as a single composition or as separate compositions. When administered separately, each therapeutic agent in the combination can be administered simultaneously or near simultaneously, or at different times. Furthermore, the therapeutic agents are administered "in combination" even if they have different dosage forms (e.g., oral capsules and intravenous), are given at different dosing intervals, one therapeutic agent is given on a fixed dosing regimen while another is titrated up, tapered, or discontinued, or each therapeutic agent in the combination is independently titrated up, tapered, or dose-up or -down, or discontinued and / or resumed during the course of a patient's treatment. When the therapeutic agents in the combination are formulated as separate compositions, in some embodiments, the separate compositions are provided together in a kit.

[0123] In certain embodiments, any of the anti-TIGIT antibodies disclosed herein are administered or applied sequentially with one or more additional active therapeutic agents, e.g., where one or more additional active therapeutic agents are administered before or after administration of an anti-TIGIT antibody according to the present disclosure. In other embodiments, the antibody is administered simultaneously with one or more additional active therapeutic agents, e.g., where the anti-TIGIT antibody is administered simultaneously or nearly simultaneously with one or more additional therapeutic agents, and the anti-TIGIT antibody and one or more additional therapeutic agents may be present in two or more separate formulations or may be combined into a single formulation (i.e., co-formulation). Regardless of whether the additional agent is administered sequentially or simultaneously with the anti-TIGIT antibody, it is considered to be administered in combination for purposes of the present disclosure.

[0124] The antibodies of the present disclosure can be used in combination with at least one other (active) agent in any manner appropriate under the circumstances. In one embodiment, treatment with at least one active agent and at least one anti-TIGIT antibody of the present disclosure is maintained for a certain period of time. In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable), while treatment with an anti-TIGIT antibody of the present disclosure is maintained at a certain dosing regimen. In a further embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable), while treatment with an anti-TIGIT antibody of the present disclosure is reduced (e.g., by reducing the dose, reducing the dosing frequency, or shortening the treatment regimen). In yet another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable), and treatment with an anti-TIGIT antibody of the present disclosure is increased (e.g., by increasing the dose, increasing the dosing frequency, or extending the treatment regimen). In yet another embodiment, treatment with at least one active agent is maintained and treatment with an anti-TIGIT antibody of the present disclosure is reduced or discontinued (e.g., dose reduced, administration frequency reduced, or treatment regimen shortened). In yet another embodiment, treatment with at least one active agent and treatment with an anti-TIGIT antibody of the present disclosure are reduced or discontinued (e.g., dose reduced, administration frequency reduced, or treatment regimen shortened).

[0125] Treatment with the antibodies of the disclosure can be combined with other treatments that are effective against the disorder being treated. When used to treat a proliferative condition, cancer, tumor, or precancerous disease, disorder, or condition, the antibodies of the disclosure can be combined with chemotherapy, radiation (e.g., local or systemic radiation therapy), stem cell therapy, surgery, or treatment with other biologics.

[0126] The antibodies of the present disclosure can be administered with a vaccine to induce an immune response against cancer. Such an immune response is enhanced by the antibodies of the present disclosure. The vaccine can include an antigen, or a fragment thereof, expressed on the surface of cancerous cells and / or tumors, optionally linked to a carrier molecule, that is effective in eliciting an immune response.

[0127] In some embodiments, one or more of the additional therapeutic agents is an immunomodulatory agent. Suitable immunomodulatory agents that can be used in the present disclosure include activating monoclonal antibodies (mAbs) against stimulatory receptors such as CD40L, B7, and B7RP1; anti-CD40, anti-CD38, anti-ICOS, and 4-IBB ligand; dendritic cell antigen loading (in vivo or in vitro); anti-cancer vaccines such as dendritic cell cancer vaccines; cytokines / chemokines such as IL1, IL2, IL12, IL18, ELC / CCL19, SLC / CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC, IFNα / β, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccharide (LPS); indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors and immunostimulatory oligonucleotides.

[0128] In certain embodiments, the present disclosure provides methods for inhibiting tumor growth, comprising administering an anti-TIGIT antibody described herein in combination with a signal transduction inhibitor (STI) to achieve additive or synergistic inhibition of tumor growth. As used herein, the term "signal transduction inhibitor" refers to an agent that selectively inhibits one or more steps in a signal transduction pathway. Signal transduction inhibitors (STIs) contemplated by the present disclosure include: (i) bcr / abl kinase inhibitors (e.g., imatinib mesylate, GLEEVEC®), (ii) epidermal growth factor (EGF) receptor inhibitors and antibodies, including kinase inhibitors (e.g., gefitinib, erlotinib, afatinib, and osimertinib), (iii) her-2 / neu receptor inhibitors (e.g., HERCEPTIN®), (iv) inhibitors of Akt family kinases or the Akt pathway (e.g., rapamycin), (v) cell cycle kinase inhibitors (e.g., flavopiridol), and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immune modulation may also be used in combination with the anti-TIGIT antibodies described herein to inhibit tumor growth in cancer patients.

[0129] In some embodiments, one or more of the additional therapeutic agents is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; ethylenimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, and chloromethylmelamine; Fosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pomalidomide, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tuberculin antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens, e.g., calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenergics, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptic acetate Titanium; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2"-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mi Tobronitol; Mitolactol; Pipobroman; Gacitosine; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel, nab-paclitaxel, and docetaxel; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum and platinum coordination complexes, such as cisplatin, carboplatin, and oxaliplatin; Vinblastine; Etoposide (VP-16); Ifos Examples of anti-inflammatory drugs include, but are not limited to, phenidate; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamycin; capecitabine; anthracyclines; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0130] Chemotherapeutic agents also include antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, ketoxifene, onapristone, and toremifene, and antiandrogens, such as abiraterone, enzalutamide, apalutamide, darolutamide, flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above, that act to regulate or inhibit hormone action on tumors. In certain embodiments, the combination therapy includes a chemotherapy regimen comprising one or more chemotherapeutic agents. In certain embodiments, the combination therapy includes the administration of a hormone or related hormonal agent.

[0131] Additional therapeutic modalities that can be used in combination with anti-TIGIT antibodies include radiation therapy, antibodies against tumor antigens, antibody-toxin conjugates, T cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy) containing TLR agonists used to stimulate such antigen-presenting cells.

[0132] In certain embodiments, the present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with RNA interference-based therapy to silence gene expression. RNAi begins with the cleavage of long double-stranded RNA into smaller interfering RNAs (siRNAs). One strand of the siRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC), which is then used to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand. RISC can either bind to or cleave the mRNA, both of which prevent translation.

[0133] In certain embodiments, the present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with agents that modulate adenosine levels. Such therapeutic agents can act on ectonucleotides, catalyzing the conversion of ATP to adenosine, including ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, ​​also known as CD39 or cluster of differentiation 39), which hydrolyzes ATP to ADP and ADP to AMP, and 5'-nucleotidase, ecto- (NT5E or 5NT, also known as CD73 or cluster of differentiation 73), which converts AMP to adenosine. In one embodiment, the present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with CD73 inhibitors, such as those described in International Publication Nos. 2017 / 120508, 2018 / 094148, and 2018 / 067424. In one embodiment, the CD73 inhibitor is AB680. Another approach targets adenosine A2a and A2b receptors. Combination with antagonists of A2a and / or A2b receptors is also contemplated. In one embodiment, the present disclosure contemplates combination with an adenosine receptor antagonist described in WO 2018 / 136700 or WO 2018 / 204661. In one embodiment, the adenosine receptor antagonist is AB928 (etremadenant).

[0134] In certain embodiments, the present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K), particularly the PI3Kγ isoform. PI3Kγ inhibitors can stimulate anti-cancer immune responses through modulation of myeloid cells, for example, by inhibiting suppressive myeloid cells, attenuating immunosuppressive tumor-infiltrating macrophages, or stimulating macrophages and dendritic cells to produce cytokines that contribute to effective T-cell responses, resulting in reduced cancer development and spread. Exemplary PI3Kγ inhibitors that can be used in combination with the anti-TIGIT antibodies described herein include those described in International Publication No. WO 2020 / 0247496(A1). In one embodiment, the PI3Kγ inhibitor is IPI-549.

[0135] In certain embodiments, the present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with inhibitors of arginase, which has been shown to cause or contribute to pro-inflammatory immune dysfunction, tumor immune escape, immunosuppression and immunopathology of infectious diseases. Representative arginase compounds can be found, for example, in International Application No. PCT / US2019 / 020507 and International Publication No. WO2020 / 102646.

[0136] In certain embodiments, the present disclosure contemplates the use of an anti-TIGIT antibody according to the present disclosure with an inhibitor of HIF-2α, which plays an important role in the cellular response to low oxygen availability. Under hypoxic conditions, hypoxia-inducible factor (HIF) transcription factors can activate the expression of genes that regulate metabolism, angiogenesis, cell proliferation and survival, immune evasion, and inflammatory responses. Overexpression of HIF-2α has been associated with poor clinical outcomes in patients with various cancers, and hypoxia is also common in many acute and chronic inflammatory diseases, such as inflammatory bowel disease and rheumatoid arthritis.

[0137] The present disclosure also contemplates the use of the anti-TIGIT antibodies described herein in combination with one or more RAS signaling inhibitors. Oncogenic mutations in RAS family genes, such as HRAS, KRAS, and NRAS, are associated with various cancers. For example, in KRAS family genes, mutations such as G12C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S, among others, have been observed in multiple tumor types. Direct and indirect inhibitory strategies have been investigated to inhibit mutant RAS signaling. Indirect inhibitors target effectors other than RAS in the RAS signaling pathway, including, but not limited to, inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORC1, SHP2 (PTPN11), and AKT. Non-limiting examples of indirect inhibitors under development include RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312, TNO155, RLY-1971, and BI1701963. Direct inhibitors of RAS mutants are also being explored, generally targeting the KRAS-GTP complex or the KRAS-GDP complex. Representative direct RAS inhibitors under development include, but are not limited to, sotorasib (AMG510), MRTX849, mRNA-5671, and ARS1620. In some embodiments, the one or more RAS signaling inhibitors are selected from the group consisting of a RAF inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a PTEN inhibitor, an SOS1 inhibitor, an mTORC1 inhibitor, a SHP2 inhibitor, and an AKT inhibitor. In other embodiments, the one or more RAS signaling inhibitors directly inhibit RAS mutants.

[0138] In some embodiments, the present disclosure is directed to the combination of an anti-TIGIT antibody according to the present disclosure with one or more inhibitors of anexelekto (i.e., AXL). The AXL signaling pathway is associated with tumor growth and metastasis and is thought to mediate resistance to various cancer therapies. There are various AXL inhibitors in development that also inhibit other kinases in the TAM family (i.e., TYRO3, MERTK), as well as other receptor tyrosine kinases such as MET, FLT3, RON, and AURORA, among others. Representative multikinase inhibitors include gilteritinib, merestinib, cabozantinib, BMS777607, and foretinib. AXL-specific inhibitors, such as SGI-7079, TP-0903 (i.e., duvelmatinib), BGB324 (i.e., bemcentinib), and DP3975, have also been developed.

[0139] In certain embodiments, the present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with adoptive cell therapy, a new and promising form of personalized immunotherapy in which immune cells with anti-tumor activity are administered to cancer patients. Adoptive cell therapy has been studied, for example, using tumor-infiltrating lymphocytes (TILs) and T cells engineered to express chimeric antigen receptors (CARs) or T cell receptors (TCRs). Adoptive cell therapy generally involves collecting T cells from an individual, genetically modifying them to target specific antigens or enhance their anti-tumor effects, expanding them to sufficient numbers, and infusing the genetically modified T cells into a cancer patient. T cells can be collected from the patient (e.g., autologous) for later reinfusion of the expanded cells, or can be collected from a donor patient (e.g., allogeneic).

[0140] T cell-mediated immunity involves multiple sequential steps, each controlled by balancing stimulatory and inhibitory signals to optimize the response. Nearly all inhibitory signals in the immune response ultimately regulate intracellular signaling pathways, but many are initiated through membrane receptors, whose ligands are either membrane-bound or soluble (cytokines). While costimulatory and inhibitory receptors and ligands that regulate T cell activation are not overexpressed in cancer compared with normal tissues, inhibitory ligands and receptors that regulate T cell effector function in tissues are generally overexpressed on tumor cells or non-transformed cells associated with the tumor microenvironment. The function of soluble and membrane-bound receptors (ligand immune checkpoints) can be modulated using agonist antibodies (costimulatory pathways) or antagonist antibodies (inhibitory pathways). Thus, in contrast to most currently approved antibodies for cancer therapy, antibodies that block or stimulate immune checkpoints do not directly target tumor cells but rather target lymphocyte receptors or their ligands to enhance intrinsic antitumor activity. [See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64].

[0141] Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated on various types of tumor cells and candidates for blockade include PD-1 (programmed cell death protein 1), PD-L1 (programmed cell death 1 ligand 1), BTLA (B and T lymphocyte attenuator), CTLA4 (cytotoxic T lymphocyte-associated antigen 4), TIM-3 (T cell immunoglobulin mucin protein 3), LAG-3 (lymphocyte-activation gene 3), TIGIT (T cell immunoreceptor with Ig and ITIM domains), and killer cell inhibitory receptors (which can be divided into two classes based on their structural features: i) killer cell immunoglobulin-like receptors (KIRs), and ii) C-type lectin receptors (members of the type II transmembrane receptor family)). Other less well-understood immune checkpoints have been described in the literature, including both receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1)) [see Pardoll, (April 2012) Nature Rev. Cancer 12:252-64].

[0142] The present disclosure contemplates the use of anti-TIGIT antibodies described herein in combination with inhibitors of the aforementioned immune checkpoint receptors and ligands, as well as those yet to be described. Certain modulators of immune checkpoints are currently approved, and many others are in development. When approved for the treatment of melanoma in 2011, the fully humanized CTLA4 monoclonal antibody ipilimumab (e.g., YERVOY®, Bristol Myers Squibb) became the first immune checkpoint inhibitor to receive regulatory approval in the United States. Fusion proteins containing CTLA4 and antibodies (CTLA4-Ig, abatcept (e.g., ORENCIA®, Bristol Myers Squibb)) have been used to treat rheumatoid arthritis, and other fusion proteins have been shown to be effective in renal transplant patients sensitized to Epstein-Barr virus. The next class of immune checkpoint inhibitors to receive regulatory approval were directed against PD-1 and its ligands, PD-L1 and PD-L2. Approved anti-PD-1 antibodies include nivolumab (e.g., OPDIVO®, Bristol Myers Squibb) and pembrolizumab (e.g., KEYTRUDA®, Merck) for a variety of cancers, including squamous cell carcinoma, classical Hodgkin lymphoma, and urothelial carcinoma. Approved anti-PD-L1 antibodies include avelumab (e.g., BAVENCIO®, EMD Serono and Pfizer), atezolizumab (e.g., TECENTRIQ®, Roche / Genentech), and durvalumab (e.g., IMFINZI®, AstraZeneca) for certain cancers, including urothelial carcinoma. In some combinations provided herein, the immune checkpoint inhibitor is selected from MEDI-0680, nivolumab, pembrolizumab, avelumab, atezolizumab, budidjalimab, BI-754091, camrelizumab, cosibelimab, durvalumab, dostallimab, cemiplimab, sintilimab, tislelizumab, toripalimab, retifanlimab, sasanlimab, and zimvelerimab (AB122).In some embodiments, the immune checkpoint inhibitor is MEDI-0680 (AMP-514, WO 2012 / 145493) or pidilizumab (CT-011). Another method for targeting the PD-1 receptor is a recombinant protein called AMP-224, which is composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1. In one embodiment, the present disclosure contemplates the use of an anti-TIGIT antibody according to the present disclosure with a PD-1 antibody. In one embodiment, the PD-1 inhibitor is dimverelimab. In some embodiments, the anti-TIGIT antibody is provided in an amount of about 200-1500 mg every three weeks, and the anti-PD-1 antibody is provided in an amount of about 100-1200 mg every three weeks. In another embodiment, the anti-TIGIT antibody described herein is given in an amount of about 300-1800 mg every four weeks and the anti-PD-1 antibody is provided in an amount of about 200-1500 mg every four weeks. In yet another embodiment, the anti-PD-1 antibody is dimvelelimab and is provided in an amount of about 360 mg or 480 mg every three or four weeks.

[0143] In another aspect, the present disclosure contemplates combination with cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-B, VEGF, and other immunosuppressive cytokines) or cytokines that stimulate T cell activation to stimulate an immune response.

[0144] In yet another aspect, T cell responses can be stimulated by a combination of the disclosed anti-TIGIT antibodies with one or more of (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, PVRIG, Galectin-9, CEACAM-1, BTLA, CD69, Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and / or (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD2. Other agents that can be used in combination with the anti-TIGIT antibodies of the present disclosure to treat cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the anti-TIGIT antibodies described herein can be used in combination with KIR antagonists, such as lirilumab.

[0145] Still other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including, but not limited to, CSF-1R antagonists such as RG7155 (WO 11 / 70024, WO 11 / 107553, WO 11 / 131407, WO 13 / 87699, WO 13 / 119716, WO 13 / 132044) or CSF-1R antagonist antibodies, including FPA-008 (WO 11 / 140249, WO 13169264, WO 14 / 036357).

[0146] In another aspect, the disclosed anti-TIGIT antibodies can be used in combination with one or more of agonistic agents that bind positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome distinct immunosuppressive pathways within the tumor microenvironment (e.g., blockade of inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), depletion or inhibition of Tregs (e.g., use of anti-CD25 monoclonal antibodies (e.g., daclizumab) or ex vivo anti-CD25 bead depletion), or reversal / prevention of T cell anergy or exhaustion), and agents that induce innate immune activation and / or inflammation at the tumor site.

[0147] In one aspect, the immuno-oncology agent is a CTLA-4 antagonist, such as an antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, ipilimumab (e.g., YERVOY®, Bristol Myers Squibb) or tremelimumab. In another aspect, the immuno-oncology agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for example, atezolizumab (MPDL3280A, WO 2010 / 077634) (e.g., TECENTRIQ®, Roche / Genentech), durvalumab (MEDI4736), BMS-936559 (WO 2007 / 005874), and MSB0010718C (WO 2013 / 79174). In another embodiment, the immuno-oncological agent is a LAG-3 antagonist, such as an antagonistic LAG-3 antibody. Suitable LAG-3 antibodies include, for example, BMS-986016 (WO 10 / 19570, WO 14 / 08218), or IMP-731 or IMP-321 (WO 8 / 132601, WO 09 / 44273). In another embodiment, the immuno-oncological agent is a CD137 (4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (WO 12 / 32433). In another embodiment, the immuno-oncological agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO 06 / 105021, WO 09 / 009116), and MK-4166 (WO 11 / 028683). In another embodiment, the immuno-oncological agent is an OX40 agonist, such as an agonist OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383 or MEDI-6469. In another embodiment, the immuno-oncological agent is an OX40L antagonist, such as an antagonistic OX40 antibody. Suitable OX40L antagonists include, for example, RG-7888 (WO 06 / 029879).In another aspect, the immuno-oncological agent is a CD40 agonist, such as an agonist CD40 antibody. In yet another embodiment, the immuno-oncological agent is a CD40 antagonist, such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, lucatamumab or dacetuzumab. In another aspect, the immuno-oncological agent is a CD27 agonist, such as an agonist CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab. In another aspect, the immuno-oncological agent is MGA271 (directed against B7H3) (WO 11 / 109400). In yet another embodiment, a combination of an anti-TIGIT antibody according to the present disclosure with an agent directed against Trop-2, such as the antibody-drug conjugate sacituzumab govitecan-hziy, is contemplated. In yet another embodiment, a combination of an anti-TIGIT antibody described herein with an agent that inhibits the CD47-SIRPα pathway is contemplated. An example of an anti-CD47 antibody is magrolimab.

[0148] Examples of therapeutic agents useful in combination therapy for the treatment of cardiovascular and / or metabolic related diseases, disorders, and conditions include statins, which inhibit the enzymatic synthesis of cholesterol (e.g., CRESTOR®, LESCOL®, LIPITOR®, MEVACOR®, PRAVACOL®, and ZOCOR®), bile acid resins, which sequester cholesterol and inhibit its absorption (e.g., COLESTID®, LO-CHOLEST®, PREVALITE®, QUESTRAN®, and WELCHOL®), and steroids, which inhibit the absorption of cholesterol (e.g., TRIMETHYL 4, TRIMETHYL 5, TRIMETHYL 6, TRIMETHYL 7, TRIMETHYL 8). These include ezetimibe (ZETIA®), which blocks cholesterol, fibric acids (e.g., TRICOR®), which can lower triglycerides and modestly increase HDL, niacin (e.g., NIACOR®), which modestly lowers LDL cholesterol and triglycerides, and / or combinations of the foregoing (e.g., VYTORIN® (ezetimibe and simvastatin)). Alternative cholesterol treatments that may be candidates for use in combination with the anti-TIGIT antibodies described herein include various supplements and herbs (e.g., garlic, policosanol, and guggul).

[0149] Examples of therapeutic agents useful in combination therapy for immune and inflammatory related diseases, disorders, or conditions include aspirin, ibuprofen, and other propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac), and acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac). Nonsteroidal anti-inflammatory drugs (NSAIDs) such as flufenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid, and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam, and tenoxicam), salicylates (acetylsalicylic acid, sulfasalazine), and pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2) inhibitors.

[0150] Other active agents for combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. Such combinations can be particularly advantageous because they can reduce or even eliminate one or more adverse effects of the steroid by tapering the required steroid dose.

[0151] For example, additional examples of active agents that can be used in combination to treat rheumatoid arthritis include cytokine suppressive anti-inflammatory drugs (CSAIDs), antibodies or antagonists to other human cytokines or growth factors, such as TNF, LT, IL-10, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, or PDGF.

[0152] Specific combinations of active agents can interfere with different points in the autoimmune and subsequent inflammatory cascade, including TNF antagonists, such as chimeric, humanized, or human TNF antibodies, REMICADE®, HUMIRA®, anti-TNF antibody fragments (e.g., CDP870), and soluble p55 or p75. TNF receptors, their derivatives, including p75TNFRIgG (ENBREL® or p55TNFR1gG (Lenercept), soluble IL-13 receptors (sIL-13), and TNFα-converting enzyme (TACE) inhibitors may also be effective, as may IL-1 inhibitors (e.g., interleukin-1 converting enzyme inhibitors). Other combinations include interleukin-11, anti-P7s, and p-selectin glycoprotein ligand (PSGL). Other examples of agents useful in combination with the A2AR / A2BR inhibitors described herein include interferon-131a (AVONEX®), interferon-131b (BETASERON®), copaxone, hyperbaric oxygen, intravenous immunoglobulin, clabribine, and antibodies or antagonists of other human cytokines or growth factors (e.g., antibodies against CD40 ligand and CD80).

[0153] In some embodiments, the combination is an antibody of the present disclosure and a second antibody directed against a surface antigen preferentially expressed on cancer cells compared to control normal tissue. Some examples of antibodies that can be administered in combination with the antibodies of the present disclosure for the treatment of cancer include Herceptin® (trastuzumab) directed against the HER2 antigen, Avastin® (bevacizumab) directed against VEGF, or antibodies directed against the EGF receptor, such as Erbitux® (cetuximab) and Vectibix® (panitumumab). Other agents that can be administered include antibodies or other inhibitors of PD-1, PD-L1, CTLA-4, 4-1BB, BTLA, PVRIG, VISTA, TIM-3, and LAG-3, or other downstream signaling inhibitors, such as mTOR and GSK3β inhibitors, and cytokines, such as interferon gamma, IL-2, and IL-15. Some specific examples of additional agents include ipilimumab, pazopanib, sunitinib, dasatinib, pembrolizumab, INCR024360, dabrafenib, trametinib, atezolizumab (MPDL3280A), erlotinib (e.g., TARCEVA®), cobimetinib, nivolumab, and zimverelimab. The choice of a second antibody or other agent for combination therapy depends on the cancer being treated. Optionally, the cancer is tested for antigen expression or preferential expression to guide the selection of an appropriate antibody. In some embodiments, the isotype of the second antibody is human IgG1 to promote effector functions such as ADCC, CDC, and phagocytosis.

[0154] Similar combination therapies can be used to treat or prevent infectious diseases, such as viral, bacterial, fungal, and parasitic infectious diseases, disorders, and conditions, and disorders related thereto. For example, the antibodies of the present disclosure can be combined with antibodies against pathogens or vaccines against pathogens, such as palivizumab against Rous sarcoma virus. The vaccine can be a protein of the pathogen or a fragment thereof that is effective in eliciting an immune response. The antibodies of the present disclosure enhance the immune response of the antibodies or vaccine against the pathogen. The antibodies of the present disclosure can also be administered with ex vivo expanded T cells or natural killer cells.

[0155] Such combination therapies include antiviral agents that target various stages of the viral life cycle and have different mechanisms of action, including, but not limited to, inhibitors of viral uncoating (e.g., amantadine and rimantidine), reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine), drugs that target integrase, drugs that block transcription factor binding to viral DNA, drugs that affect translation (e.g., antisense molecules) (e.g., fomivirsen), drugs that modulate translation / ribozyme function, protease inhibitors, viral assembly modulators (e.g., rifampicin), antiretroviral agents such as nucleoside reverse transcriptase inhibitors (e.g., azidothymidine (AZT), ddl, ddC, 3TC, d4T), non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine), nucleoside reverse transcriptase inhibitors, and drugs that prevent viral particle release (e.g., zanamivir and oseltamivir). Treatment and / or prevention of certain viral infections (eg, HIV) often involves a group (a "cocktail") of antiviral agents.

[0156] Other antiviral agents contemplated for use in combination with any of the anti-TIGIT antibodies disclosed herein include abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, ATRIPLA®, boceprevirert, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, These include, but are not limited to, flucloxil, inosine, various interferons (e.g., pegylated interferon alfa-2a), lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nexavir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, TRUVADA®, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

[0157] The present disclosure contemplates the use of any of the anti-TIGIT antibodies disclosed herein in combination with an antiparasitic agent. Such agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, bithionol, oxamniquine, metrifonate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox, and nitroimidazole. Those skilled in the art will recognize other agents that may find utility in treating parasitic disorders.

[0158] Embodiments of the present disclosure contemplate the use of any of the anti-TIGIT antibodies disclosed herein in combination with an agent useful for treating or preventing bacterial disorders. Antibacterial agents can be classified in various ways based on their mechanism of action, chemical structure, and spectrum of activity. Examples of antibacterial agents include those that target bacterial cell walls (e.g., cephalosporins and penicillins) or cell membranes (e.g., polymyxins), or those that interfere with essential bacterial enzymes (e.g., sulfonamides, rifamycins, and quinolines). Most antibacterial agents that target protein synthesis (e.g., tetracyclines and macrolides) are bacteriostatic, while agents such as aminoglycosides are bactericidal. Another means of classifying antibacterial agents is based on their target specificity: "narrow-spectrum" agents target specific types of bacteria (e.g., gram-positive bacteria such as streptococci), while "broad-spectrum" agents have activity against a wider range of bacteria. Those skilled in the art will recognize the types of antibacterial agents that are appropriate for use in specific bacterial infections.

[0159] Embodiments of the present disclosure contemplate the use of any of the anti-TIGIT antibodies disclosed herein in combination with an agent useful for the treatment or prevention of fungal disorders. Antifungal agents include polyenes (e.g., amphotericin, nystatin, and pimaricin), azoles (e.g., fluconazole, itraconazole, and ketoconazole), allylamines (e.g., naftifine and terbinafine) and morpholines (e.g., amorolfine), and antimetabolites (e.g., 5-fluorocytosine).

[0160] The present disclosure includes pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0161] V. Other Uses The anti-TIGIT antibodies of the present disclosure can be used to detect TIGIT in clinical diagnostic or therapeutic contexts or in research. For example, the antibodies can be used to detect the presence of TIGIT on T cells, natural killer cells, and cancer cells as an indicator that a subject is suffering from a cancer or infectious disease amenable to treatment. Expression of TIGIT on T cells, natural killer cells, and / or cancer cells in a subject suffering from cancer or an infectious disease also provides an indication that the cancer or infectious disease is amenable to treatment with the antibodies of the present disclosure. The antibodies can also be sold as research reagents for laboratory studies in detecting T cells, natural killer cells, and cancer cells and their responses to various stimuli. For such uses, the antibodies can be labeled with one or more detectable signals, including, but not limited to, fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in the form of a kit with all the necessary reagents for performing an assay for TIGIT. The anti-TIGIT antibodies of the present disclosure can also be used to purify TIGIT, for example, by affinity chromatography.

[0162] VI. Kit An antibody against TIGIT can be combined with any of the second antibodies or agents described for use in combination therapy as a component of a kit. The present disclosure provides one or more kits containing one or more of the antibodies disclosed herein and one or more pharmaceutically acceptable excipients or carriers (including, but not limited to, phosphate-buffered saline solution, water, sterile water, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers, and various types of wetting agents). Additives such as alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like can also be included in the kits of the present disclosure along with the carriers, diluents, or excipients. In one embodiment, pharmaceutically acceptable carriers suitable for use in the antibody compositions disclosed herein are sterile, pathogen-free, and / or safe for administration to subjects without the risk of associated infection and other undue harmful side effects. In the kit, each agent may be provided in a separate vial with instructions for administration after combination or instructions for separate administration. The kit may also include written instructions for the proper handling and storage of any of the anti-TIGIT antibodies disclosed herein.

[0163] VII. Embodiments Embodiment 1. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising: (a) a heavy chain (HC) complementarity-determining region (CDR) 1 having at least 80% sequence identity to SEQ ID NO: 36, a HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 37, and a HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 38; and a light chain (LC) CDR1 having at least 80% sequence identity to SEQ ID NO: 39, a light chain (LC) CDR2 having at least 80% sequence identity to SEQ ID NO: 40, and a light chain (LC) CDR3 having at least 80% sequence identity to SEQ ID NO: 41. (b) a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 42, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 43, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 44; and (c) a light chain variable region comprising an LC-CDR1, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 46, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 47; (d) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 48, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 49, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 50; and (d) a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 52, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 53, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 54, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 55, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 56;and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 57, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 58, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 59; (e) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 60, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 61, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 64, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 60, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 66, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 69, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 55, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 71, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65; (h) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 72, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 73, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67;and a light chain variable region comprising an LC-CDR1 having at least 80% sequence identity to SEQ ID NO: 63, an LC-CDR2 having at least 80% sequence identity to SEQ ID NO: 68, and an LC-CDR3 having at least 80% sequence identity to SEQ ID NO: 65, or (i) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity to SEQ ID NO: 74, an HC-CDR2 having at least 80% sequence identity to SEQ ID NO: 75, and an HC-CDR3 having at least 80% sequence identity to SEQ ID NO: 67; and an anti-TIGIT antibody or antigen-binding fragment thereof comprising:

[0164] Embodiment 2. (a) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 36, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 37, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 38; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising identity to SEQ ID NO: 39, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 40, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 41; (b) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 42, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 43, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 44; and a light chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 45, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 46, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 47; (c) an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 48, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 49. a heavy chain variable region comprising an HC-CDR1 having an amino sequence comprising SEQ ID NO: 51, an LC-CDR2 having an amino sequence comprising SEQ ID NO: 52, and an LC-CDR3 having an amino sequence comprising SEQ ID NO: 53; (d) a heavy chain variable region comprising an HC-CDR1 having an amino sequence comprising SEQ ID NO: 54, an HC-CDR2 having an amino sequence comprising SEQ ID NO: 55, and an HC-CDR3 having an amino sequence comprising SEQ ID NO: 56; and a light chain variable region comprising an LC-CDR1 having an amino sequence comprising SEQ ID NO: 57, an LC-CDR2 having an amino sequence comprising SEQ ID NO: 58, and an LC-CDR3 having an amino sequence comprising SEQ ID NO: 59; (e) a heavy chain variable region comprising an HC-CDR1 having an amino sequence comprising SEQ ID NO: 60, an HC-CDR2 having an amino sequence comprising SEQ ID NO: 61, and an HC-CDR3 having an amino sequence comprising SEQ ID NO: 62;and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 64, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 66, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 69, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 55, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 70; and a LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 71, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65. the anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 1, comprising: (h) a heavy chain variable region comprising an HC-CDR1 having an amino sequence comprising SEQ ID NO: 72, an HC-CDR2 having an amino sequence comprising SEQ ID NO: 73, and an HC-CDR3 having an amino sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino sequence comprising SEQ ID NO: 65; or (i) a heavy chain variable region comprising an HC-CDR1 having an amino sequence comprising SEQ ID NO: 74, an HC-CDR2 having an amino sequence comprising SEQ ID NO: 75, and an HC-CDR3 having an amino sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino sequence comprising SEQ ID NO: 65.

[0165] Embodiment 3. (a) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:1 and a light chain variable region having at least 80% sequence identity to SEQ ID NO:2; (b) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:3 and a light chain variable region having at least 80% sequence identity to SEQ ID NO:4; (c) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:5 and a light chain variable region having at least 80% sequence identity to SEQ ID NO:6; (d) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO:7 % sequence identity to SEQ ID NO: 8, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 8; (e) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 9, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 10; (f) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 11, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12; (g) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 13, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 14; (h) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 15 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 16; (i) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 17 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 12; (j) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 76 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 77. (k) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 78 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 77; (l) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 76 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 79; or (m) a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 78 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 79.The anti-TIGIT antibody or antigen-binding fragment thereof according to embodiment 1 or 2.

[0166] Embodiment 4. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0167] Embodiment 5. The anti-TIGIT antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 4, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric, humanized, or veneered antibody.

[0168] Embodiment 6. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 5, wherein the chimeric antibody comprises a human IgG1 / kappa Fab constant domain.

[0169] Embodiment 7. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a human antibody.

[0170] Embodiment 8. The anti-TIGIT antibody or antigen-binding fragment thereof inhibits binding of TIGIT to CD155, and optionally, the anti-TIGIT antibody or antigen-binding fragment thereof has an IC of about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, measured as in Example 1. 50 The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein the antibody inhibits binding by

[0171] Embodiment 9. The anti-TIGIT antibody or antigen-binding fragment thereof of any one of embodiments 1 to 5 or 7 to 8, wherein the antibody further comprises a mutant heavy chain constant region selected from mutant human IgG1, mutant human IgG2, mutant human IgG3, or mutant human IgG4, and optionally a human light chain constant region.

[0172] Embodiment 10. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 9, wherein the variant heavy chain constant region has an enhanced or reduced effector function compared to the wild-type heavy chain constant region.

[0173] Embodiment 11. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 10, wherein the variant human IgG heavy chain constant region comprises SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 101.

[0174] Embodiment 12. The anti-TIGIT antibody or antigen-binding fragment thereof of any one of embodiments 1 to 5 or 7 to 8, wherein the antibody further comprises a wild-type human IgG heavy chain constant region and, optionally, a human light chain constant region.

[0175] Embodiment 13. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 12, wherein the wild-type human IgG heavy chain constant region comprises SEQ ID NO: 94.

[0176] Embodiment 14. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising a human light chain kappa constant region, optionally wherein the human light chain constant region comprises SEQ ID NO: 95.

[0177] Embodiment 15. An anti-TIGIT antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5 or 7 to 8, wherein the antibody has a heavy chain and a light chain, and (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 92 and the light chain has an amino acid sequence comprising SEQ ID NO: 93, or (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 96 and the light chain has an amino acid sequence comprising SEQ ID NO: 93, or (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 98 and the light chain has an amino acid sequence comprising SEQ ID NO: 93, or (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 100 and the light chain has an amino acid sequence comprising SEQ ID NO: 93.

[0178] Embodiment 16. The antibody or binding fragment thereof has (a) a denaturation constant of about 0.01 x 10 as measured by surface plasmon resonance. -11 M ~ approx. 100×10 -11 M, approx. 0.1×10 -11M ~ approx. 100×10 -11 M, approx. 0.1×10 -11 M ~ approx. 10×10 -11 M, about 1 x 10 -11 M ~ approx. 100×10 -11 M or approximately 1 x 10 -11 M ~ approx. 10×10 -11 M; (b) blocks the binding of soluble human CD155 ligand to cell-surface human TIGIT with a half maximal inhibitory concentration (IC50) of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.6 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured as in Example 1; (c) binds to at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; 82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80; or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80; or (d) any combination of (a), (b), and (c).

[0179] Embodiment 17. An anti-TIGIT antibody or antigen-binding fragment thereof described in embodiment 16, which antibody or antigen-binding fragment thereof competes for binding to TIGIT with the antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 17.

[0180] Embodiment 18. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 16 or 17, wherein the excess antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when measured in a competitive binding assay, and the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

[0181] Embodiment 19. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

[0182] Embodiment 20. A method for inhibiting the binding of TIGIT to CD155, comprising contacting TIGIT with an anti-TIGIT antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 19.

[0183] Embodiment 21. A method of treating a subject infected with a pathogen, comprising administering to the subject an effective regimen or a therapeutically effective amount of an antibody of any one of embodiments 1 to 20.

[0184] Embodiment 22. The method of embodiment 21, wherein the pathogen is a virus, bacterium, fungus, or protozoan.

[0185] Embodiment 23. The method of embodiment 22, wherein the pathogen is HIV, SIV, hepatitis, herpesvirus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, chlamydia, rickettsia, mycobacteria, staphylococci, treptocci, pneumococci, meningococci, conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospira, and lyme burgdorferi.

[0186] Embodiment 24 The method of any one of embodiments 21-23, wherein the subject is treated with a vaccine that induces an antibody-boosted immune response against the pathogen.

[0187] Embodiment 25. The method of embodiment 24, wherein the vaccine comprises a protein or fragment thereof of a pathogen.

[0188] Embodiment 26. The method of any one of embodiments 21 to 25, wherein the subject is further administered a second antibody against the pathogen, and the effector-mediated cytotoxicity of the second antibody against the pathogen is enhanced by the antibody.

[0189] Embodiment 27. The method of any one of embodiments 21-26, wherein the subject is further administered one or more of an antiviral agent, an antiparasitic agent, an antibacterial agent, or an antifungal agent.

[0190] Embodiment 28. A method for treating or effectively preventing cancer, comprising administering to a subject having or at risk of cancer an effective regimen or therapeutically effective amount of any one of the anti-TIGIT antibodies or antigen-binding fragments thereof described in any one of embodiments 1 to 27.

[0191] Embodiment 29. The method of embodiment 28, wherein the cancer is a hematological malignancy, a solid tumor, Merkel cell carcinoma, urothelial carcinoma, squamous cell carcinoma of the head and neck, B-cell lymphoma, uterine cancer, cervical cancer, testicular cancer, gastrointestinal cancer, bladder cancer, bone cancer, bone marrow, skin cancer, gallbladder cancer, heart cancer, lung cancer, salivary gland cancer, adrenal cancer, thyroid cancer, ganglionic cancer, cancer of the central nervous system (CNS) and peripheral nervous system (PNS), and cancer of the hematopoietic system, cancer of the immune system.

[0192] Embodiment 30. The method of embodiment 28 or 29, wherein the subject is administered tumor-infiltrating T cells activated by the antibody or antigen-binding fragment thereof.

[0193] Embodiment 31. The method of any one of embodiments 28-30, wherein the subject is administered a vaccine that induces an immune response against cancer, which is boosted by an antibody or antigen-binding fragment thereof.

[0194] Embodiment 32. The method of embodiment 31, wherein the vaccine comprises an antigen or a fragment thereof expressed on the surface of a cancer cell.

[0195] Embodiment 33. The method of any one of embodiments 28 to 32, wherein the subject is administered natural killer cells whose cytotoxicity against the cancer is enhanced by the antibody or antigen-binding fragment thereof.

[0196] Embodiment 34. The method of any one of embodiments 28 to 33, wherein the subject is further administered a second antibody against an antigen expressed on the surface of cells of the cancer, whereby the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the antibody or antigen-binding fragment thereof.

[0197] Embodiment 35. The method of any one of embodiments 28 to 33, wherein the subject is further administered a second antibody against an antigen expressed on the surface of an immune cell.

[0198] Embodiment 36. The method of embodiment 35, wherein the immune cells are T cells or natural killer cells.

[0199] Embodiment 37. The method of embodiment 35 or 36, wherein the antigen is CTLA-4, PD-1, or PD-L1.

[0200] Embodiment 38. The method of any one of embodiments 28 to 37, wherein the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapy, and surgery.

[0201] Embodiment 39. The method of any one of embodiments 28 to 38, wherein the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands.

[0202] Embodiment 40. The one or more immune checkpoint receptors or ligands are CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A 2a R,A 2b R.A. 2a / A 2b 40. The method of embodiment 39, wherein the antibody is selected from the group consisting of R, arginase, CD39, CD73, IDO, and TDO.

[0203] Embodiment 41. The method of embodiment 39, wherein the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimvelerimab, durvalumab, and atezolizumab.

[0204] Embodiment 42. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19 and a pharmaceutically acceptable carrier.

[0205] Embodiment 43. An anti-TIGIT antibody or antigen-binding fragment thereof that binds to an epitope of human TIGIT that includes at least one of the following amino acid residues of SEQ ID NO: 80: T55, Q56, N58, E60, D72, S80, and K82.

[0206] Embodiment 44. The antibody or antigen-binding fragment thereof binds to at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80, and optionally 44. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 43, which binds to an epitope comprising, selectively, at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80, (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80, or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80.

[0207] Embodiment 45. An anti-TIGIT antibody or antigen-binding fragment thereof described in embodiment 43 or 44, wherein the antibody or antigen-binding fragment thereof competes for binding to TIGIT with the antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 19.

[0208] Embodiment 46. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 43 or 44, wherein the excess antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when measured in a competitive binding assay, and the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

[0209] Embodiment 47. An anti-TIGIT antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 19, wherein the antibody or antigen-binding fragment thereof binds to an epitope of human TIGIT that includes at least one of the following amino acid residues of SEQ ID NO: 80: T55, Q56, N58, E60, D72, S80, and K82.

[0210] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0211] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, the version associated with the accession number as of the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or, if applicable, the filing date of the priority application referencing the accession number. Similarly, where different versions of a publication, website, etc. are published at different times, the version most recently published as of the effective filing date of this application is meant unless otherwise specified. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other, unless otherwise specified.

[0212] Although the invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. [Example]

[0213] The following examples discuss the generation, characterization, and humanization of antibodies against human TIGIT, and also provide representative methods by which the binding characteristics of the antibodies described in this application can be determined.

[0214] Example 1. Generation of anti-TIGIT antibodies Anti-TIGIT antibodies were obtained from immunized mice. The extracellular domains of His-tagged human TIGIT protein (hTIGIT-His) with SEQ ID NO: 83 and cynoTIGIT protein (cTIGIT-His) with SEQ ID NO: 85 were transiently expressed in HEK293 cells and purified by anti-His affinity chromatography. BALB / c mice were immunized with a mixture of recombinant hTIGIT-His and cTIGIT-His proteins via RIMMS. Plasma titers were tested before the final booster immunization by ELISA assay against the immunogen, and successful titers were confirmed. After the final booster immunization, peripheral blood was collected along with the spleen, inguinal, brachial, axillary, and cervical lymph nodes. The collected material was subjected to B cell purification and then fused to generate hybridomas for initial screening.

[0215] Primary screening of hybridomas was performed using an ELISA assay 10 days after fusion. 384-well ELISA plates were coated with 1 μg / mL hTIGIT-His protein and blocked. 20 μL of hybridoma supernatant was added and allowed to bind to the TIGIT-coated plate. After incubation at room temperature, the plate was washed, and antibodies bound to the TIGIT-coated plate were detected using an HRP-conjugated goat anti-mouse IgG antibody.

[0216] Positive hybridoma cells were then grown in 48-well plates, and supernatants were collected to test antibody specificity in an ELISA assay. ELISA plates were coated with hTIGIT-His protein or cTIGIT-His protein, and CD47-His protein (Acro Biosystems, catalog no. CD7-H5227) was used as a control counterassay to deselect antibodies that recognize the His tag of the immunogen protein. Antibodies that showed positive binding to both human and cynoTIGIT but negative binding to the human-tagged control protein were then tested for functional blocking of TIGIT / CD155 binding.

[0217] The human TIGIT extracellular domain was fused to a mouse Fc sequence, and the resulting hTIGIT-mFc (SEQ ID NO: 87) protein was expressed in HEK293 cells and purified by protein A affinity chromatography. A blocking assay for CD155 / TIGIT interaction was established using recombinant human CD155 (hCD155-hFc) containing the extracellular domain fused to a human Fc sequence from R&D Systems (catalog no. 9174-CD-01M). To test the functional blocking activity of the antibodies, ELISA plates were coated with 0.5 μg / mL of hTIGIT-mFc protein. After blocking, hybridoma supernatants were added together with 0.5 μg / mL of hCD155-hFc protein. After incubation, the ELISA plates were washed, and bound hCD155-hFc was detected using an HRP-conjugated goat anti-human IgG antibody. Clones were identified that were able to bind to both human and cynomolgus-TIGIT and whose antibody binding was able to block the CD155 / TIGIT interaction.

[0218] These clones were further expanded, and antibodies were purified using a protein G column. These purified antibodies were tested by flow cytometry for binding to human and cynoTIGIT expressed on the cell surface. Stable CHO-K1 cell lines expressing full-length human TIGIT clone 2A7 (Swiss-Prot Q495A1, SEQ ID NO: 80) or full-length cynoTIGIT clone C10 (Swiss-Prot A0A2K5UW92, SEQ ID NO: 84) were developed. For flow assays, cells were harvested and incubated in 100 μL of HBSS buffer in the presence (or absence) of various concentrations of antibody at 4°C for 1 hour. After washing with HBSS, antibody binding on the cells was detected with 2 μg / mL Alexa488-conjugated goat anti-mouse IgG antibody (ThermoFisher Scientific, Catalog No. A-11001) for 30 minutes at 4°C. Cells were then washed, resuspended in PBS, and subjected to flow cytometry using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA). The geometric mean of fluorescence intensity was obtained for a single whole-cell population. Antibodies capable of binding to both human and cynoTIGIT expressed on the cell surface were further tested for their blocking activity against recombinant human CD155 binding to human TIGIT on the surface of CHO cells. CHO-hTIGIT cells (10 5 Cells) were incubated with 2.5 μg / mL of hCD155-Fc protein in the presence of various concentrations of antibody for 1 hour at room temperature. After washing with HBSS buffer, hCD155-Fc binding to hTIGIT-CHO cells was detected with PerCP-eFluor 710-conjugated anti-CD155 antibody (ThermoFisher, Cat. No. 1550-42). Cells were then washed and subjected to flow cytometry analysis. Table 4 shows the median effective concentrations (EC ) of antibodies binding to human and cynoTIGIT. 50 ) (n=2), and IC for inhibition of CD155 binding to TIGIT 50 Shows. [Table 4]

[0219] The top hybridoma cell lines were selected based on their binding affinity to both human and cynoTIGIT and their ability to block CD155 binding to TIGIT. These clonal hybridomas were expanded, and the heavy and light chain variable regions (VH and VL, respectively) of the murine anti-TIGIT antibodies were sequenced according to standard procedures. The mature VH and VL amino acid sequences of antibodies 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28O12 are shown in Figures 1A-1I, with the CDRs underlined. The CDR sequence assignments and amino acid position numbering follow the Kabat definition.

[0220] Seven of the antibodies shown in Figures 1A-1I were recombinantly expressed as mouse-human chimeras with mouse variable domains and human IgG1 / kappa constant domains. Recombinant proteins were expressed in HEK293 cells and purified by protein A affinity chromatography.

[0221] The binding ability of these chimeric anti-TIGIT antibodies to cell surface-overexpressed human and cynoTIGIT was confirmed using the flow cytometry assay described above. After incubation of the chimeric anti-TIGIT antibodies with hTIGIT-CHO or cTIGIT-CHO, bound antibodies were detected using Alexa 488-conjugated goat anti-human IgG antibody (ThermoFisher Scientific, Cat. No. A-11013). The antibody functional activity inhibiting recombinant hCD155-hFc binding to hTIGIT-CHO-K1 cells was also determined using the flow assay described above. EC values ​​of the anti-TIGIT chimeric antibodies binding to human and cynoTIGIT were 50 , and IC for inhibition of hCD155 binding to hTIGIT-expressing cells 50 was determined and is shown in Table 5. [Table 5]

[0222] Isolated human CD4 + and CD8 + The binding ability of the chimeric anti-TIGIT antibody to endogenously expressed TIGIT on human CD4 cells was tested using a flow cytometry assay. + or CD8 + T cells were analyzed using RosetteSep™ human CD4 + T cell enrichment cocktail (Stemcell, Cat. No. 15022) or human CD8 + T cell enrichment cocktail (Stemcell, Cat. No. 15022) was used to isolate ECs from human whole blood. As shown in Table 6, comparable ECs were isolated from human whole blood. 50 However, human CD4 + or CD8 + The binding affinity of recombinant anti-TIGIT antibodies to cells was similar to that of overexpressed full-length human TIGIT on CHO-K1 cells, although differences in maximum binding activity (MFImax) were observed between clones. [Table 6]

[0223] The binding of recombinant anti-TIGIT chimeric antibodies to cynomolgus monkey whole blood was tested and CD4 + and CD8 +The antibody's binding ability to endogenous cynoTIGIT protein on cells was confirmed. Cynomolgus monkey whole blood was incubated with recombinant anti-TIGIT chimeric antibody at 20 μg / mL, 5 μg / mL, 1 μg / mL, and 0.2 μg / mL. After 30 minutes of incubation at 4°C, RBC lysis was performed at room temperature for 15 minutes. Cells were then washed, collected by centrifugation, and blocked with a cocktail containing Fc block (BD Biosciences, Cat. No. 564219) and Live-dead fixable Aqua (Invitrogen, Cat. No. L34957). Cell-bound anti-TIGIT antibodies were detected with anti-human IgGFc-biotin (Southern Biotech, Cat. No. 9040-08) for 30 minutes at 4°C, followed by washing and centrifugation of the cells and a second incubation with PE-conjugated streptavidin (Invitrogen, Cat. No. 12-4317-87) for 30 minutes at 4°C. Wild-type human IgG1 antibody was used as an isotype control, and directly conjugated anti-human TIGIT-PE (eBiosciences, Cat. No. 12-9500-42) was used as a positive control. Figure 2 shows that Ch24F8, Ch28O12, and Ch22B22 bind to cynomolgus monkey CD4 + and CD8 + The results show that the antibody was able to bind to cynoTIGIT expressed on cells. The geometric mean fluorescence intensity (gMFI) was obtained, and the data were expressed as fold gMFI relative to the isotype control.

[0224] The kinetic binding of these recombinant anti-TIGIT antibodies to human TIGIT was determined by surface plasmon resonance (SPR) using a Bio-Rad ProteOn XPR36 instrument. The recombinant antibodies were immobilized using a protein A-coated GLC sensor chip, and soluble His-tagged TIGIT (Acro Biosystems, catalog number TIT-H52H3) was used as the analyte. Binding constants were determined at 25°C. As shown in Table 7, among the seven recombinant anti-TIGIT chimeric antibodies tested, clone 24F8 had the highest equilibrium dissociation constant (K D ) has the highest binding affinity. [Table 7]

[0225] Example 2. Generation of humanized anti-TIGIT antibodies The murine antibody 24F8 was selected for humanization using CDR grafting technology (Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989). The murine variable heavy (VH) and variable light (VL) chain sequences of 24F8 were used to identify the two closest human germline sequences for each chain. For VH, the IGHV4-34 antibody shared 70% sequence identity with the IGHV4-34 antibody. * 09, and IGHV4-4, which has 66% identity * 02 was found. For VL, IGKV1-33 had 70% sequence identity. * 01, and IGKV3-15, which has 67% identity * 01 was found (Table 8). [Table 8]

[0226] The positioning of the three heavy chain CDR (HC-CDR) sequences in the VH chain and the three light chain CDR (LC-CDR) sequences in the VL chain was defined according to Kabat.

[0227] Human acceptors for the VH and VL frameworks were searched in the GenBank database (Benson et al., Nucleic Acids Res. 2005, 33, D34-D38) and human cDNA-encoded VH and VL sequences were identified (see Table 8).

[0228] CDR grafting for each human acceptor was performed using HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50) for the VH acceptor, and LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53) for the VL acceptor. The resulting sequences were inspected for any potential sites for introduction of post-translational modifications or chemical degradation to confirm their absence. Putative residues for murine backmutation were also identified using antibody homology graphical modeling.

[0229] For the human acceptor grafted with two VHs and two VLs, oligonucleotides were designed and synthesized as Fab fragments with human IgG1 / kappa constant domains and inserted into a vector system for high-throughput screening of expression levels and biophysical properties without the need for protein purification (Zhang & Hirama, U.S. Patent Application Publication No. 2012 / 0178110). All four combinations of VH1 (SEQ ID NO: 76) or VH2 (SEQ ID NO: 78) with VL1 (SEQ ID NO: 79) or VL2 (SEQ ID NO: 77) (Figures 1J-1M) were screened together with chimeric Fab mVH+mVL constructed from mouse 24F8VH (SEQ ID NO: 5) and 24F8VL (SEQ ID NO: 6) domains and human IgG1 / kappa Fab constant domains. The supernatants of Fab secreted as SASA (single domain antibody to serum albumin) fusion proteins were analyzed by surface plasmon resonance (SPR) using a Biacore 8K instrument with Fab capture using a BSA-coated chip and soluble His-tagged TIGIT as the analyte. Kinetic binding data for human and cynoTIGIT are shown in Table 9 for the five Fab fragments. [Table 9]

[0230] These data demonstrate that the VH1+VL2 and VH2+VL2 humanized variable domain combinations have similar equilibrium dissociation constants (K D), and retained most of the binding affinity of the mouse / human chimeric Fab. As a result, constructs containing backmutations of murine framework residues were not considered. These two VH / VL combinations (Figure 1J and Figure 1K) were selected for construct design of the full-length Hu24F8.1 and Hu24F8.2 IgG1 / kappa antibodies, respectively.

[0231] Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA are IgG1 / kappa antibodies having a heavy chain constant region having the amino acid sequence of SEQ ID NO:97 and a light chain constant region having the amino acid sequence of SEQ ID NO:95. The designation "IgG1.AA" indicates that the heavy chain constant region has leucine to alanine amino acid substitutions at positions 234 and 235 (Eu numbering). In contrast, the designation "IgG1" indicates a wild-type IgG1 Fc region. For example, Hu24F8.2-IgG1 is an IgG1 / kappa antibody having a heavy chain constant region having the amino acid sequence of SEQ ID NO:94 and a light chain constant region having the amino acid sequence of SEQ ID NO:95. The Hu24F8.1-IgG1.AA, Hu24F8.2-IgG1.AA, and Hu24F8.2-IgG1 antibodies used in these examples were recombinantly produced in HEK293 or CHO cells and purified by Protein A affinity chromatography.

[0232] Purified full-length antibodies Hu24F8.1-IgG1.AA (containing VH1+VL2) and Hu24F8.2-IgG1.AA (containing VH2+VL2), along with a full-length chimeric antibody of 24F8 mouse VH+VL and human IgG1 / kappa constant domains (Ch24F8), were analyzed by SPR on a Biacore T200 instrument by antibody capture using an anti-human IgG-coated chip and soluble hTIGIT-His or cTIGIT-His as the analyte. Kinetic binding data for human and cynoTIGIT are shown in Table 10. [Table 10]

[0233] Dynamic binding affinity data for both full-length humanized antibodies confirmed that the binding affinity of the murine antibody was fully retained, eliminating the need to introduce backmutations of any murine framework residues.

[0234] Example 3. In vitro binding studies of anti-TIGIT antibodies The binding of antibodies Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA to TIGIT expressed on the cell surface was examined by flow cytometry. Cells expressing TIGIT on the cell surface were harvested and incubated in 100 μL of HBSS buffer in the presence (or absence) of various concentrations of antibody for 1 hour at 4°C. After washing with HBSS, antibody binding on the cells was detected with 2 μg / mL Alexa488-labeled goat anti-human IgG antibody (ThermoFisher Scientific, catalog number A-11013) for 30 minutes at 4°C. The cells were then washed, resuspended in PBS, and subjected to flow cytometry using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA). The geometric mean of fluorescence intensity was obtained for a single whole-cell population. In the case of transient transfection, untransfected cells were used to gate on the positively bound cell population to obtain the percentage of positive cells. Data were calculated with GraphPad Prism using standard four-parameter curve fitting.

[0235] Using stable CHO-K1 cell lines expressing human TIGIT (clone 2A7) and cynoTIGIT (clone C10), Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA were tested for binding to human TIGIT and cynoTIGIT, respectively, as described above. Hu24F8.1-IgG1.AA had an EC 50 on human TIGIT (Figure 3A), with an EC of 0.237 ± 0.33 nM (n = 6). 50Hu24F8.2-IgG1.AA bound to cynoTIGIT with an EC of 0.29 ± 0.15 nM (n = 8) (Figure 3B). 50 on human TIGIT (Figure 3A), with an EC of 0.35 ± 0.16 nM (n = 6). 50 These results demonstrate that both Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA strongly bind to human and cynoTIGIT expressed on the cell surface.

[0236] Binding of Hu24F8.2-IgG1.AA to mouse TIGIT (Swiss-Prot Q86176, SEQ ID NO: 88) and rat TIGIT (Swiss-Prot D3ZTQ2, SEQ ID NO: 89) was examined using CHO-K1 cells transiently transfected with full-length mouse or rat TIGIT expression constructs. Mouse TIGIT expression was confirmed using the control antibody GNE10A7 (U.S. Patent No. 9,499,596, Clark et al., 2016), and rat TIGIT expression was confirmed using the control antibody eBioscience™ G1GD7 (Invitrogen, Catalog No. 12-9501-82). When tested with up to 30 nM antibody, Hu24F8.2-IgG1.AA does not bind to either mouse TIGIT (Figure 4A) or rat TIGIT (Figure 4B).

[0237] Hu24F8.2-IgG1.AA isolated human CD8 + Binding to CD8 T cells was examined by flow cytometry. + T cells were cultured in RosetteSep™ human CD8 T cells according to the manufacturer's recommendations. + T cell enrichment cocktail (Stemcell, Cat. No. 15023) was used to isolate cells. Cells were then activated with anti-CD3 / CD28 beads supplemented with 20 U / mL rhIL-2 for 7-9 days. Activated or non-activated CD8 +The cells were blocked with human Fc block (BD Biosciences, Cat. No. 564219) and then subjected to flow cytometry antibody binding assays. As shown in Figures 5A and 5B, Hu24F8.2-IgG1.AA bound to either non-activated or CD8 cells. + EC of 0.098 ± 0.013 nM (n = 2) for cells 50 and activated CD8 + Binding to cells was 0.14 ± 0.036 nM (n = 2), respectively. EC 50 Activated and non-activated CD8 + Although the binding signals were similar across cells, there were significant differences in the maximum binding signals, which was due to the CD8 + This is consistent with increased TIGIT expression in

[0238] Example 4. In vitro blocking studies of anti-TIGIT antibodies The ability of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA to block the interaction of TIGIT with CD155 was analyzed by flow cytometry using CHO cells stably overexpressing human TIGIT (CHO-hTIGIT) and human CD155-Fc fusion recombinant soluble protein (hCD155-Fc), as described in Example 1. As shown in Figure 6, both Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA dose-dependently blocked the interaction between hCD155-Fc on the cell surface and CHO-hTIGIT. The IC 50 was 0.68 nM for Hu24F8.1-IgG1.AA and 0.67 nM for Hu24F8.2-IgG1.AA.

[0239] Example 5. Characterization of anti-TIGIT antibodies using Jurkat Dual Reporter Cell Line The functional activity of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA in blocking the human TIGIT receptor was assayed using Promega's TIGIT / CD155 Blockade Bioassay (Promega, Catalog No. J2205). In this assay, the effector cell line was a Jurkat cell line overexpressing TIGIT and carrying a luciferase reporter that is activated downstream of the T cell receptor (TCR). Another stable cell line was a CHO-K1 cell line overexpressing human CD155 in addition to T cell activation proteins that bind to and activate the TCR. This CD155 aAPC / CHO-K1 cell line served as artificial antigen-presenting cells. When these two cell lines were cocultured, TCR activation by the aAPC on the CHO-K1 cells led to activation of the reporter construct, and pathway activation was inhibited by the TIGIT / CD155 interaction, resulting in a low luciferase signal. The presence of anti-TIGIT antibodies inhibits the TIGIT / CD155 interaction, resulting in a TIGIT inhibitory effect and an increased luciferase signal. The assay was performed according to the manufacturer's protocol. Briefly, effector Jurkat cells were allowed to recover overnight in a 96-well plate in a cell culture incubator. A serial dilution of the test antibody was added to the effector cells, followed by antigen-presenting CD155 aAPC / CHO-K1 cells. After 6 hours of co-culture at 37°C and 5% CO2, Bio-Glo reagent, a luciferase substrate, was added, and the luminescence signal was read using Envision (PerkinElmer). As shown in Figure 7, Hu24F8.1-IgG1.AA had an EC of 3.35 ± 0.26 nM (n = 3). 50 could enhance reporter activity in a dose-dependent manner, while Hu24F8.2-IgG1.AA had an EC 50 The human IgG1 control did not show any effect.

[0240] Example 6. Molecular analysis of anti-TIGIT antibodies Expression, purification, and crystallization of TIGIT and Fab A soluble protein of the mature extracellular domain of human TIGIT (residues 22-130) was recombinantly expressed in HEK293 cells. The construct (SEQ ID NO: 90) contained a C-terminal hexahistidine tag with a (Gly)4-Ala-(Gly)4 linker, and asparagine residues 32 and 101 were mutated to glutamine to remove N-glycosylation sites. The clarified supernatant was purified by affinity chromatography using a Nickel Sepharose Excel (GE Healthcare Life Sciences) column, followed by size-exclusion chromatography (SEC) using a Superdex 200pg (GE Healthcare Life Sciences) column. TIGIT protein at a concentration of 8.4 mg / mL was in a final buffer formulation of 20 mM Tris pH 7.0, 100 mM NaCl and flash-frozen in liquid nitrogen.

[0241] Soluble Fab fragments (Fab24F8) of the human IgG1 antibody Hu24F8.2-IgG1.AA were prepared by papain (Thermo Scientific, catalog no. 20341) digestion in phosphate-buffered saline at 37°C for 3 hours, followed by overnight digestion at room temperature. The cleaved Fc fragment was removed using a MabSelect SuRe Protein A (GE Healthcare Life Sciences) column, and the flow-through was further purified by SEC using a Superdex 200pg (GE Healthcare Life Sciences) column. Fab24F8 protein at a concentration of 28 mg / mL was in a final buffer formulation of 20 mM Tris pH 7.0, 100 mM NaCl and flash-frozen in liquid nitrogen.

[0242] TIGIT was mixed with Fab24F8 protein at a 1:1 molar ratio with stirring at 4 °C for 60 min to form the Fab-TIGIT complex, followed by final SEC purification using a Superdex 200 pg (GE Healthcare Life Sciences) column, resulting in a protein eluate concentration of 44 mg / mL. The purified complex was used for crystallization studies at 20 °C using a standard screen of approximately 1500 different conditions. Initially obtained conditions were optimized using standard strategies, systematically varying parameters that significantly affected crystallization. These conditions were further refined by systematically varying pH or precipitant concentration. Crystals of the Fab-TIGIT complex suitable for structure elucidation were obtained using the sitting-drop vapor diffusion method by mixing 0.1 μL of protein solution (15 mg / mL in 20 mM Tris pH 7.0, 100 mM NaCl) with 0.1 μL of reservoir solution (20% (w / v) PEG3350, 0.20 M LiSO4).

[0243] Data collection and structural analysis The crystals were flash-frozen and measured at a temperature of 100 K. X-ray diffraction data were collected from crystals of the Fab-TIGIT complex at the Canadian Light Source (CLS, Saskatoon, Canada) using cryogenic conditions. The crystals belong to the space group P1. The data were processed using the computer software programs autoPROC, XDS, and AIMLESS (The CCP4 Suite: Programs for Protein Crystallography "Acta Cryst. D50, 760-763), see Table 11. [Table 11] 1 The value in parentheses refers to the highest resolution bin.

[0244] The phase information necessary for structure determination and analysis was obtained by molecular replacement. Previously solved structures of Fab (Bohrmann et al., J. Alzheimers Dis. 28:49-69, 2012) and TIGIT (Stengel et al., Proc. Natl. Acad. Sci. USA, 2012) were used as search models. Three molecules of the Fab-TIGIT complex are present in the crystallographic asymmetric unit. Subsequent model building and refinement were performed according to standard protocols using the programs COOT and CCP4, respectively. For calculation of the free R-factor, a measure for cross-validating the accuracy of the final model, approximately 4.6% of the measured reflections were excluded from the refinement procedure. Performing TLS refinement (using the CCP4 program REFMAC5) yielded lower R-factors and higher-quality electron density maps. Automatically generated local NCS restraints were applied. The water model was constructed using the "Find waters" algorithm in COOT by placing water molecules at peaks in the Fo-Fc map contoured at 3.0σ, then refined with REFMAC5 and checked all waters with the validation tool in COOT. The criteria for listing suspect waters were a B-factor of 80 Å. 2 The data in the resolution range of 73.5 to 2.24 Å were in the final cycle of refinement and R cryst and R free The R-factors for were 22.3 and 26.8%, respectively. A Ramachandran plot of the final model shows that 89.8% of all residues are in the most favored regions, 8.8% in the additionally allowed regions, and 0.7% in the generally accepted regions. See Table 12 for a summary of the refinement. [Table 12] 1 The test set contains 4.6% of the measured reflections 2 Mean square deviation from geometric target 3Calculations by MOLEMAN 4 Calculation by PROCHECK Structure of Fab24F8 bound to human TIGIT

[0245] The three independent molecules of the complex are in the crystallographic asymmetric unit, and their atomic coordinates are pairwise superimposable with a root-mean-square deviation of 0.53 to 1.13 Å for all non-hydrogen atoms. The final model includes residues Gln1 to Ser223 of the Fab heavy chain, Glu1 to Cys214 of the Fab light chain, and Met22 to Ser129 of TIGIT. Several short loop regions are not fully defined by the electron density and are not included in the final model.

[0246] The HC-CDR2 & HC-CDR3 of the Fab heavy chain and all three LC-CDRs of the light chain are integrated into the large β-sheet structure of TIGIT, i.e., the polypeptide chain of SEQ ID NO: 80. 55 TQVNWEQQDQLLAICNADLGWHISPSFK 82 and 109 IYH 111 The Fab fragment forms extensive interactions with β-strands C, C', C'' and F, and loops C'C'' and C''D (Figures 8 and 9). This binding interaction is 790±10 Å between the Fab fragment and TIGIT (PISA, EMBL-EBI). 2 This results in a protein-protein interface with a surface area of ​​1000 kJ / s (n=3). The molecular nature of this interaction is both hydrophilic and hydrophobic. TIGIT residues Thr55, Gln56, Asn58, Glu60, Asp72, Ser80, and Lys82 (shown as sticks in Figure 9) form direct or water-mediated hydrogen-bonding interactions (donor / acceptor interatomic distances of 3.1 Å or less) with residues of the Fab heavy and light chain CDRs. Furthermore, TIGIT residue Glu60 forms a salt bridge with Arg30 on the Fab light chain (Table 13). TIGIT hydrophobic residues Leu65, Ile68, Leu73, Pro79, and Ile109 (shown as boxes in Figure 9) make van der Waals contacts with residues of the Fab heavy and light chains. [Table 13] * Water-mediated hydrogen bonding

[0247] Figure 10A shows a schematic diagram of the complex structure of the N-terminal Ig-like domain (SEQ ID NO: 91) of human CD155 (ribbon-shaped) bound to human TIGIT (represented as a molecular surface), as reported in Stengel et al., 2012. Figure 10B shows a superposition of CD155 in the same orientation as in Figure 10A on a schematic diagram of the crystal structure complex of Fab24F8 (each represented by a molecular surface) bound to TIGIT. It can be clearly demonstrated that Hu24F8.2 or other antibodies derived from the murine antibody 24F8, which bind to the extracellular domain of TIGIT, block the binding of CD155 to TIGIT.

[0248] Example 7. Anti-TIGIT antibodies enhance T cell responses both alone and in combination with anti-PD-1 antibodies This example demonstrates that Hu24F8.2-IgG1, alone or in combination with an anti-PD-1 antibody such as AB122, enhances human primary T cell responses in healthy or cancer subjects. PBMCs from healthy and cancer subjects treated with 0.1, 1, or 10 μg / mL of Hu24F8.2-IgG1 had significantly increased IL-2 concentrations compared to isotype controls. PBMCs from healthy subjects treated with a combination of 10 μg / mL of Hu24F8.2-IgG1 and 1 μg / mL of an anti-human PD-1 antibody (AB122, dimvelelimab) had significantly higher IL-2 levels compared to AB122 alone.

[0249] PBMCs from healthy subjects were isolated from Leukoreduction System (LRS) chambers, and PBMCs from cancer subjects were isolated from CPT tubes and cultured in vitro with either 0.1, 1, or 10 μg / mL Hu24F8.2, 1 μg / mL AB122, or a combination of 10 μg / mL Hu24F8.2-IgG1 and 1 μg / mL AB122 in the presence of 1 ng / mL SEA. After 4 days, IL-2 concentrations in the supernatants were measured by cytometric bead array (CBA). IgG1 isotype was included as a negative control.

[0250] method PBMCs from healthy subjects were isolated from the LRS chamber, while PBMCs from cancer subjects were isolated from the CPT tube. PBMCs were collected at 2 x 10 per mL. 6 Cells were resuspended at a concentration of 0.1, 1, and 10 μg / mL and 100 μL was dispensed per well into a 96-well round-bottom plate. Four-fold concentrated antibodies, resuspended in 50 μL of CTS Optimizer medium per well, were added to the appropriate wells: Hu24F8.2-IgG1 or human IgG1 isotype control, were added to final concentrations of 0.1, 1, and 10 μg / mL, and AB122 or human IgG4 isotype control, were added to a final concentration of 1 μg / mL. The assay plate was incubated at 37°C and 5% CO2 for 1 hour. Four-fold concentrated Staphylococcal enterotoxin A (SEA), resuspended in 50 μL of CTS Optimizer medium per well, was added to the appropriate wells at a final concentration of 1 ng / mL. The assay plate, with a final well volume of 200 μL, was incubated at 37°C and 5% CO2 for 4 days, after which the supernatant was collected for subsequent quantification of secreted IL-2. The supernatant was diluted 1:2 in the assay diluent of the Human Soluble Protein Master Buffer Kit. Assay performance, data acquisition, and quantification were performed using the Human IL-2 Flex Set with the Human Soluble Protein Master Buffer Kit according to the manufacturer's instructions.

[0251] resultPBMCs from all donors used in this study were confirmed to express TIGIT on the CD4+ T cell population, a non-T regulatory cell, which responds to SEA stimulation of CD14+ monocytes and CD155 (TIGIT's ligand). PBMCs isolated from 10 healthy subjects and 7 cancer subjects were cultured with 0.1, 1, or 10 μg / mL Hu24F8.2-IgG1 alone, 1 μg / mL AB122 alone, or a combination of 10 μg / mL Hu24F8.2-IgG1 and 1 μg / mL AB122 in the presence of 1 ng / mL SEA. IL-2 concentrations were measured in the supernatants after 4 days. IL-2 levels in the Hu24F8.2-treated groups were compared with those in the respective IgG1 isotype control-treated groups, while IL-2 levels in the AB122 and Hu24F8.2-IgG1 combination-treated groups were compared with those in the AB122-only treatment group (Tables 14 and 15). An example of IL-2 levels for one healthy subject (#566) is shown in Figure 11 for all concentrations of Hu24F8.2-IgG1 tested. For these two subjects, there was a statistically significant increase in IL-2 secretion at all concentrations of Hu24F8.2-IgG1 tested compared with the respective IgG1 isotype controls. Healthy subject #566 also showed a statistically significant increase in IL-2 secretion with AB122 and Hu24F8.2-IgG1 combination treatment compared with AB122 treatment alone. At the cohort level, 10 μg / mL Hu24F8.2-IgG1 treatment resulted in a statistically significant increase in IL-2 secretion compared to the IgG1 isotype control in PBMCs from both healthy and cancer subjects (Figure 12A), and combined treatment with AB122 and Hu24F8.2-IgG1 resulted in a statistically significant increase in IL-2 secretion compared to AB122 treatment alone in PBMCs from healthy subjects (Figure 12B).In summary, 0.1 μg / mL Hu24F8.2-IgG1 significantly increased IL-2 concentrations (relative to the isotype) in PBMC samples from 6 / 10 healthy subjects (1.1-3.4 fold) and in PBMC samples from 2 / 5 cancer subjects (1.6-1.7 fold), whereas 1 μg / mL Hu24F8.2-IgG1 increased IL-2 concentrations in PBMC samples from 7 / 10 healthy subjects (1.4-4.0 fold) and in PBMC samples from 4 / 7 cancer subjects (1.6-2.0 fold). Hu24F8.2-IgG1 at 10 μg / mL significantly increased (vs. isotype) IL-2 concentrations in PBMC samples from 7 / 10 healthy subjects (1.2-4.0-fold) and in PBMC samples from 4 / 7 cancer subjects (1.3-2.0-fold), and Hu24F8.2-IgG1+AB122 at 10 μg / mL increased IL-2 concentrations in PBMC samples from 6 / 10 healthy subjects (1.9-8.3-fold) (vs. AB122 alone). [Table 14] a n=3 number of experiments b One-way ANOVA with Sidak's multiple comparison test (isotype vs. Hu24F8.2, or AB122 + Hu24F8.2 vs. AB122); NS, not significant; nt, not tested; * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001. [Table 15] a n=3 Number of experiments b One-way ANOVA with Sidak's multiple comparison test (isotype vs. Hu24F8.2); NS, not significant; nt, not tested; * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001

[0252] These data demonstrate that Hu24F8.2-IgG1, alone or in combination with an anti-PD-1 antibody such as AB122, enhances human primary T cell responses in healthy or cancer subjects.

[0253] Example 8. Antibody characterization by in vitro complement-dependent cytotoxicity (CDC) assay CDC is an immune response in which the complement system participates in antibody-dependent cell killing through the binding of complement component 1q (C1q) to the fragment crystallizable (Fc) region of an antibody. This initiates the complement cascade reaction, leading to the formation of a membrane attack complex that damages the cell membrane of target cells expressing the target protein recognized by the Fab region of the antibody. In this example, the CDC activity of Hu24F8.2-IgG1 was confirmed using GS-J1 / TIGIT cells (GenScript M00693) in the presence of normal human serum complement (NHSC, Quidel).

[0254] Cell lysis by CDC was determined using the Cell Titer-Glo® Assay Kit (Promega), which determines the number of viable cells in culture based on ATP quantification. Briefly, GS-J1 / TIGIT cells (5000 cells / well) were treated with 10 μg / mL of Hu24F8.2-IgG1 or human IgG1 (Abcam) negative control in the presence of 5%, 10%, or 20% NHSC for 4 hours at 37°C and 5% CO2 (NHSC% optimization assay), or GS-J1 / TIGIT cells (5000 cells / well) were treated with serially diluted Hu24F8.2-IgG1 (10 μg / mL) or human IgG1 (10 μg / mL) in the presence of 5% NHSC for 4 hours at 37°C and 5% CO2 (CDC concentration-response test). As a positive control, Raji cells (ATCC CCL-86, 5000 cells / well) were treated with serially diluted rituximab (10 μg / mL) in the presence of 5% NHSC for 4 hours at 37°C and 5% CO2. After incubation with the test or control antibody, Cell Titer-Glo® reagent was added, the samples were incubated for an additional 10-30 minutes at room temperature, and luminescence was read on a PHERAstar FSX (BMG LabTech). Cell lysis by CDC was calculated using the following formula: % Cell Lysis = 100% × (1-(RLU) sample -RLU NHSC ) / (RLU cell+NHSC -RLU NHSC )).

[0255] The results of the system control (rituximab on Raji cells) met quality control standards in both studies. However, no concentration-dependent CDC activity of the test sample (Hu24F8.2-IgG1) or negative control (human IgG1) was observed in either the NHSC% optimization assay or the CDC concentration-response study. The results are summarized in Tables 16 and 17, respectively. FACS analysis of Hu24F8.2-IgG1 binding to GS-J1 / TIGIT cells was used to confirm that the lack of CDC was not due to lack of binding to the target cells (data not shown). [Table 16] [Table 17]

[0256] Example 9. Antibody characterization by SPR Hu24F8.2-IgG1 was analyzed by SPR using a BioRad ProteOn XPR36 instrument with antibody capture using anti-human IgG-coated or Protein A-coated chips at six different densities. The analyte was soluble hTIGIT-His diluted to 33 nM (stock solution prepared at 33.3 μM) as the highest concentration and tested in triplicate in a 3-fold dilution series on the Hu24F8.2-IgG1 surface. The running buffer contained 10 mM HEPES, 150 mM NaCl, 0.05% Tween®-20, and 0.2 mg / mL BSA. All data were collected at 25°C. Data from all six surface densities were globally fit to a 1:1 interaction model using local Rmax. The results are shown in Table 18. [Table 18] NOTE: Numbers in parentheses represent standard errors in the last reported digits from the global fits of six different density surfaces.

Claims

1. An anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to human TIGIT, (a) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 48, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 49, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 50; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 51, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 52, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 53; (b) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 42, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 43, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 44; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 45, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 46, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 47; (c) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 54, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 55, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 56; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 57, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 58, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO:

59. (d) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 61, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 64, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO:

65. (e) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 66, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO:

65. (f) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO:69, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO:55, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO:70; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO:71, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO:68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO:65; (g) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 72, an HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 73, and an HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65; or (h) an anti-TIGIT antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HC-CDR1 having an amino sequence comprising SEQ ID NO: 74, HC-CDR2 having an amino sequence comprising SEQ ID NO: 75, and HC-CDR3 having an amino sequence comprising SEQ ID NO: 67; and a light chain variable region comprising LC-CDR1 having an amino sequence comprising SEQ ID NO: 63, LC-CDR2 having an amino sequence comprising SEQ ID NO: 68, and LC-CDR3 having an amino sequence comprising SEQ ID NO:

65. (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:78, and a light chain variable region having at least 90% sequence identity to SEQ ID NO:77; (b) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:5, and a light chain variable region having at least 90% sequence identity to SEQ ID NO:6; (c) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 76, and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 77; (d) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 76 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 79; or (e) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 78, and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 79; (f) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:3, and a light chain variable region having at least 90% sequence identity to SEQ ID NO:4; (g) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:7, and a light chain variable region having at least 90% sequence identity to SEQ ID NO:8; (h) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 9, and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 10; (i) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 11, and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 12; (j) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 13, and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 14; (k) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 15, and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 16; (l) an anti-TIGIT antibody or its antigen-binding fragment according to claim 1, comprising a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 17 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 12;

3. An anti-TIGIT antibody or an antigen-binding fragment thereof described in claim 1 or 2, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof is a monoclonal antibody.

4. An anti-TIGIT antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a veneered antibody.

5. An anti-TIGIT antibody or its antigen-binding fragment described in claim 4, wherein the chimeric antibody comprises a human IgG1 / kappa Fab constant domain.

6. An anti-TIGIT antibody or an antigen-binding fragment thereof described in claim 1 or 2, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof is a human antibody.

7. An anti-TIGIT antibody or an antigen-binding fragment thereof described in any one of claims 1 to 6, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof inhibits binding of TIGIT to CD155.

8. An anti-TIGIT antibody or its antigen-binding fragment described in any one of claims 1 to 4 or 6 to 7, wherein the antibody further comprises a mutant heavy chain constant region selected from mutant human IgG1, mutant human IgG2, mutant human IgG3, or mutant human IgG4.

9. An anti-TIGIT antibody or its antigen-binding fragment described in claim 8, wherein the mutant heavy chain constant region has an enhanced or reduced effector function compared to the wild-type heavy chain constant region.

10. An anti-TIGIT antibody or its antigen-binding fragment described in Claim 9, wherein the mutant human IgG heavy chain constant region comprises SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO:

101.

11. An anti-TIGIT antibody or its antigen-binding fragment described in any one of claims 1 to 4 or 6 to 7, wherein the antibody further comprises a wild-type heavy chain constant region.

12. An anti-TIGIT antibody or its antigen-binding fragment described in Claim 11, wherein the wild-type heavy chain constant region comprises sequence number 94.

13. An anti-TIGIT antibody or antigen-binding fragment thereof described in claim 11 or 12, comprising a human light chain kappa constant region.

14. The antibody having a heavy chain and a light chain, (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 92 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 96 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 98 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (d) An anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or 6 to 7, wherein the heavy chain has an amino acid sequence comprising SEQ ID NO: 100 and the light chain has an amino acid sequence comprising SEQ ID NO:

93.

15. The antibody or binding fragment thereof, (a) has an equilibrium binding constant (KD) of about 0.01×10 −11 M to about 100×10 −11 M, as measured by surface plasmon resonance, where about means within ±10% of the value provided; (b) blocks the binding of soluble human CD155 ligand to cell-surface human TIGIT with a half maximal inhibitory concentration (IC50) of about 0.2 nM to about 2 nM, where about means within ±10% of the value provided; (c) binds to an epitope that includes at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80, and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80; (iv) E60, D72 and K82 of SEQ ID NO: 80, or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80; or (d) The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, which is any combination of (a), (b), and (c).

16. An anti-TIGIT antibody or antigen-binding fragment thereof described in claim 15, wherein the antibody or antigen-binding fragment thereof competes for binding to TIGIT with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 15.

17. An anti-TIGIT antibody or antigen-binding fragment thereof described in claim 15 or 16, wherein an excess of the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55% when measured in a competitive binding assay, where about means within ±10% of the value provided, and the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO:

93.

18. A composition for use in a method for inhibiting the binding of TIGIT to CD155, comprising an anti-TIGIT antibody or antigen-binding fragment thereof described in any one of claims 1 to 17, said method comprising contacting TIGIT with said anti-TIGIT antibody or antigen-binding fragment thereof.

19. A composition for the treatment or effective prevention of cancer in a subject having or at risk of cancer, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof described in any one of claims 1 to 18.

20. The composition described in claim 19, wherein the cancer is a hematological malignancy, a solid tumor, Merkel cell carcinoma, urothelial cancer, squamous cell carcinoma of the head and neck, B-cell lymphoma, uterine cancer, cervical cancer, testicular cancer, gastrointestinal cancer, bladder cancer, bone cancer, bone marrow, skin cancer, gallbladder cancer, heart cancer, lung cancer, salivary gland cancer, adrenal cancer, thyroid cancer, ganglionic cancer, cancer of the central nervous system (CNS) and peripheral nervous system (PNS), as well as cancer of the hematopoietic system and cancer of the immune system.

21. The composition described in claim 19 or 20, wherein the subject is administered tumor-infiltrating T cells activated by the antibody or its antigen-binding fragment.

22. A composition described in any one of claims 19 to 21, wherein the subject is administered a vaccine that induces an immune response against the cancer, which is enhanced by the antibody or its antigen-binding fragment.

23. The composition of claim 22, wherein the vaccine comprises an antigen or a fragment thereof expressed on the surface of a cancer cell.

24. A composition described in any one of claims 19 to 23, wherein the subject is administered natural killer cells whose cytotoxicity against the cancer is enhanced by the antibody or its antigen-binding fragment.

25. The composition described in any one of claims 19 to 24, wherein the subject is further administered a second antibody against an antigen expressed on the surface of cancer cells, whereby the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the antibody or its antigen-binding fragment.

26. A composition described in any one of claims 19 to 25, wherein the subject is further administered a second antibody against an antigen expressed on the surface of an immune cell.

27. ​​The composition described in claim 26, wherein the immune cells are T cells or natural killer cells.

28. The composition described in claim 25 or 26, wherein the antigen is CTLA-4, PD-1 or PD-L1.

29. The composition described in any one of claims 19 to 28, wherein the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapy, and surgery.

30. A composition described in any one of claims 19 to 29, wherein the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands.

31. The composition of claim 30, wherein the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A2aR, A2bR, A2a / A2bR, arginase, CD39, CD73, IDO, and TDO.

32. The composition of claim 30, wherein the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimvelerimab, durvalumab, and atezolizumab.

33. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

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