Tigit antibodies and uses thereof

Abstract

The invention discloses an anti-human TIGIT antibody or an antigen binding fragment thereof, the anti-human TIGIT antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises antigen determining regions VH CDR1, VH CDR2 and VH CDR3, and the VL comprises antigen determining regions VL CDR1, VL CDR2 and VL CDR3. The humanized anti-human TIGIT antibody can be specifically combined with TIGIT, and shows a good clinical anti-tumor application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to an anti-TIGIT humanized antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] Although immune checkpoint inhibitors targeting PD-1 have greatly improved cancer treatment and made "advanced cancer incurable" a thing of the past, PD-1 drugs still have the problem of low efficacy, and immune-related adverse reactions often occur during use, and even lead to death. Non-small cell lung cancer and esophageal cancer are one of the main fields of PD-L1 drugs, but there are still problems such as low efficacy, which are also the main battlefields of PD-L1 inhibitors. Therefore, people are also looking for new immune checkpoints for the treatment of cancer, and TIGIT is one of the closest to clinical application. At present, TIGIT is considered to be one of the most promising and potential targets, and a variety of evidence supports that TIGIT plays a key role in limiting the adaptive immunity and innate immunity of tumors: (https: / / www.xianjichina.com / special / detail_520678.html)

[0003] TIGIT is a new immune checkpoint molecule that is expressed on the surface of two major anti-cancer cells, T cells and NK cells (Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8+T cell effector function[J]. Cancer cell, 2014, 26(6): 923-937.).

[0004] TIGIT functional axis and ligands

[0005] TIGIT stands for T cell immunoreceptor with Ig and ITIM domains (also known as WUCAM, Vstm3, VSIG9). It is a receptor of the Ig superfamily and plays a key role in limiting adaptive and innate immunity. TIGIT participates in a complex regulatory network involving multiple IRs (e.g., CD96 / TACTILE, CD112R / PVRIG), a competitive co-stimulatory receptor (DNAM-1 / CD226), and multiple ligands (e.g., CD155, CD112). TIGIT binds to CD155 with a higher affinity than the competing receptors CD226 and CD96. TIGIT binds weakly to CD112, and CD112R binds to CD112 with a higher affinity than CD226, which is a binding mode in which inhibitory receptors and co-stimulatory receptors compete to bind to the same ligand. TIGIT inhibits T cell activation by competing with CD226 and binding to PVR, disrupting CD226 activation, and other mechanisms. TIGIT is highly expressed in tumor-infiltrating T cells in multiple cancer types (Original link: https: / / www.xianjichina.com / special / detail_520678.html TIGIT is expressed by activated CD8+ T and CD4+ T cells, natural killer (NK) cells, regulatory T cells (Tregs), and follicular helper T cells, as well as on monocytes, dendritic cells (DCs), and many non-hematopoietic cells, including tumor cells of different histological types.

[0006] TIGIT structure and signaling pathway

[0007] TIGIT consists of an extracellular immunoglobulin (Ig) variable domain, a type 1 transmembrane domain, and an intracellular domain with two inhibitory motifs conserved in mice and humans: the immunoreceptor tyrosine-based inhibitory motif (ITIM) and the Ig tail tyrosine-like (ITT) motif. The crystal structure of TIGIT bound to CD155 shows that two TIGIT / CD155 dimers form a heterotetramer with the TIGIT / TIGIT cis homodimer as the core, with each TIGIT molecule binding to one CD155 molecule. This cis-trans receptor aggregation mediates cell adhesion and signaling.

[0008] In the human NK cell line YTS, TIGIT / CD155 binding initiates a major inhibitory signal through an ITT-like motif, while the ITIM motif mediates a minor inhibitory signal. Following activation by TIGIT / CD155 binding, the ITT-like motif is phosphorylated at Tyr225 and binds to the intracytoplasmic signaling molecules Grb2 and β-arrestin 2 to recruit inositol-containing SH2 phosphatase-1 (SHIP-1). SHIP-1 impedes phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. SHIP-1 also inhibits the activation of TRAF6 and NF-κB, resulting in reduced IFN-γ production by NK cells (see Fig.25 ).

[0009] TIGIT potentially suppresses innate and adaptive immunity through multiple mechanisms

[0010] First, in mouse models, TIGIT indirectly impedes T cell function by binding to CD155 on DCs. Second, TIGIT directly exhibits intrinsic inhibitory effects on immune cells. Third, multiple lines of evidence indicate that TIGIT impedes CD155-mediated CD226 activation. Fourth, the balance of TIGIT / CD226 expression regulates the effector functions of T cells and NK cells and enhances T cell-mediated tumor rejection. Fifth, TIGIT acts on Tregs to enhance immunosuppressive function and stability. Compared with TIGIT-Tregs, TIGIT+Tregs upregulated many Treg gene markers in the periphery and tumor sites. After TIGIT activation, TIGIT++Tregs produced IL-10 and fibrinogen-like protein 2 to mediate T cell inhibition.

[0011] In preclinical trials, TIGIT inhibitors have inhibitory effects on a variety of tumors and can produce synergistic effects with PD-1 drugs to enhance anti-cancer capabilities (Ge Z, Peppelenbosch MP, Sprengers D, et al. TIGIT, the next step towards successful combination immune checkpoint therapy in cancer[J]. Frontiers in Immunology, 2021, 12).

[0012] CD112R blockade, alone or in combination with TIGIT blockade and / or PD-1 blockade, can increase the ability of TILs to produce cytokines in ovarian tumors, endometriomas, and lung tumors (Whelan S, Ophir E, Kotturi MF, et al..PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+T-cell Function. Cancer Immunol Res 2019; 7:257–68).

[0013] The synergistic effect between TIGIT inhibitors and PD-L1 inhibitors, anti-TIGIT antibody drugs as new immune checkpoint antibody drugs have broad application prospects and can be used for tumor immunotherapy. Let TIGIT inhibitors become a hope for further improving the immunotherapy effect of non-small cell lung cancer and esophageal squamous cell carcinoma. At present, targeting the TIGIT-PVR pathway is becoming more and more important, and some biotechnology / pharmaceutical companies are committed to developing antibodies or bispecific antibodies against TIGIT. As of the end of 2021, a total of 23 monoclonal antibodies or bispecific antibodies against TIGIT are being commercially developed and are in different stages of clinical development. Existing clinical information records show that TIGIT is an important target for the treatment of non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical tumors, multiple myeloma, non-Hodgkin's lymphoma, B lymphocytic tumors, and plasma cell cancer. In particular, Roche's Tiragolumab has been tested in multiple cancer types such as non-small cell lung cancer and esophageal cancer. The trial has reached the Phase III stage and has been recognized as a "breakthrough therapy" by the FDA. At present, many companies including BMS, Roche, Innovent, BeiGene, etc. have conducted a number of clinical trials on monoclonal antibodies targeting TIGIT.

[0014] However, since the best treatment for targets in this pathway has not yet been commercialized, as research on the mechanism of action of the TIGIT target continues to deepen, there is still a serious unmet medical need for the development of TIGIT antibodies with new structures and new mechanisms of action resulting from new structures. Summary of the invention

[0015] In view of the deficiencies of the prior art and actual needs, the purpose of the present invention is to provide new anti-TIGIT antibodies, including nucleic acids encoding such antibodies and compositions containing such antibodies, as well as their use in enhancing anti-tumor immunity and tumor treatment. Surprisingly, it was found that the anti-TIGIT antibodies of the present invention have a high level of binding affinity to human TIGIT; in addition, these antibodies can not only block the interaction between human TIGIT and human PVR (CD155), but also block the interaction between the corresponding cynomolgus monkey proteins, specifically relieve the immunosuppressive effect of TIGIT on the body, and activate T lymphocytes, which is of great significance in improving the activity of immune cells and enhancing immune responses, preventing and treating tumors and infectious diseases.

[0016] In a first aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region (HCDRs) combination consisting of HCDR 1, HCDR 2 and HCDR3 and a light chain complementary determining region (LCDRs) combination consisting of LCDR 1, LCDR 2 and LCDR 3, wherein the heavy chain variable region comprises one, two, or more of the following CDRs:

[0017] (a) HCDR 1, which comprises the amino acid sequence shown in SEQ ID NO: 1, 7 or 13, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 1, 7 or 13;

[0018] (b) HCDR 2, which comprises the amino acid sequence shown in SEQ ID NO: 2, 8 or 14, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 2, 8 or 14;

[0019] (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, 9 or 15, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 3, 9 or 15;

[0020] In one embodiment, the light chain variable region comprises one, two, or more of the following CDRs:

[0021] (d) LCDR 1, which comprises the amino acid sequence shown in SEQ ID NO: 4, 10 or 16, or an amino acid sequence having at least 85% sequence homology to the amino acid sequence shown in SEQ ID NO: 4, 10 or 16;

[0022] (e) LCDR 2, which comprises the amino acid sequence shown in SEQ ID NO 5, 11 or 17, or an amino acid sequence having at least 85% sequence homology to 5, 11 or 17;

[0023] (f) LCDR 3, whose sequence comprises the amino acid sequence shown in SEQ ID NO: 6, 12 or 18; or an amino acid sequence having at least 85% sequence homology to SEQ ID NO: 6, 12 or 18.

[0024] In a second aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein, according to the IMGT numbering system,

[0025] The heavy chain variable region comprises HCDR1-HCDR3 as shown in SEQ ID NOs: 1-3, respectively, and the light chain variable region of the anti-TIGIT antibody comprises LCDR1-LCDR3 as shown in SEQ ID NOs: 4-6, respectively; or

[0026] The heavy chain variable region comprises HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 7-8, respectively, and the light chain variable region of the anti-TIGIT antibody comprises LCDR1-LCDR3 as shown in SEQ ID NOs: 9-12; or

[0027] The heavy chain variable region comprises HCDR1-HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 13-15, respectively, and the light chain variable region of the anti-TIGIT antibody comprises LCDR1-LCDR3 as shown in SEQ ID NOs: 16-18.

[0028] In one embodiment, the heavy chain variable region further comprises variable region heavy chain framework sequences HC-FR1, HC-FR2, HC-FR3 and HC-FR4 located between the HCDRs, thereby forming a heavy chain variable region (VH) comprising the sequence structure of the following formula:

[0029] (HC-FR1)-(HCDR1)-(HC-FR2)-(HCDR2)-(HC-FR3)-(HCDR3)-(HC-FR4).

[0030] In one embodiment, the heavy chain framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

[0031] In another embodiment, the heavy chain framework sequence is a kappa light chain sequence.

[0032] In one embodiment, the heavy chain framework region (HC-FR) sequence comprises one or more of HC-FR1, HC-FR2, HC-FR3 and HC-FR4 as described below:

[0033] The HC-FR1 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 191-104, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 91-104;

[0034] The HC-FR2 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 105-113, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 105-113

[0035] The HC-FR3 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 114-124, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 114-124

[0036] The HC-FR4 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 125-5, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 125-133, 183.

[0037] In one embodiment, the light chain further comprises a variable region light chain framework sequence LC-FR1, LC-FR2, LC-FR3 and LC-FR4 located between the LCDRs, thereby forming a light chain variable region (VL) having the following sequence structure:

[0038] (LC-FR1)-(LCDR1)-(LC-FR2)-(LCDR2)-(LC-FR3)-(LCDR3)-(LC-FR4).

[0039] In one embodiment, the light chain framework sequence is derived from a human consensus framework sequence or a human germline framework sequence.

[0040] In another embodiment, the light chain framework sequence is a lambda light chain sequence.

[0041] In one embodiment, the light chain framework sequence comprises one or more of LC-FR1, LC-FR2, LC-FR3 and LC-FR4 as described below:

[0042] The LC-FR1 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 136-150, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 136-150;

[0043] The LC-FR2 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 151-165, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 151-5;

[0044] The LC-FR3 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 166-180, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 166-180;

[0045] The LC-FR4 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 181-185, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 181-185.

[0046] In one embodiment, the heavy chain variable region of the anti-TIGIT antibody or antigen-binding fragment thereof comprises one or more framework sequences located between the VHCDRs as follows:

[0047] (HC-FR1)-(VHCDR1)-(HC-FR2)-(VHCDR2)-(HC-FR3)-(VHCDE3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences located between the VLCDRs as follows:

[0048] (LC-FR1)-(VLCDR11)-(LC-FR2)-(VLCDR2)-(LC-FR3)-(VLCDR3)-(LC-FR4), where,

[0049] The HC-FR1 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 191-104;

[0050] The HC-FR2 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 105-113,

[0051] The HC-FR3 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 114-124,

[0052] The HC-FR4 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 125-135;

[0053] or

[0054] The LC-FR1 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 136-150,

[0055] The LC-FR2 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 151-165,

[0056] The LC-FR3 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 166-180,

[0057] The LC-FR4 sequence is the amino acid sequence shown in any one of SEQ ID NOs: 181-185.

[0058] In a third aspect, the present invention provides an isolated antibody or an antigen-binding portion thereof that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23, 25 or 27, or an amino acid sequence as shown in any one of SEQ ID NOs: 29-42, or has a sequence homology of not less than 85% with an amino acid sequence as shown in any one of SEQ ID NOs: 23, 25 or 27, or an amino acid sequence as shown in any one of SEQ ID NOs: 29-42, and / or

[0059] The light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 26 or 28, or the amino acid sequence shown in any one of SEQ ID NOs: 43-56, or has a sequence homology of not less than 85% with the amino acid sequence shown in any one of SEQ ID NOs: 24, 26 or 28, or the amino acid sequence shown in any one of SEQ ID NOs: 43-56.

[0060] In one embodiment, the VH comprises an amino acid sequence selected from any one of SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27; or the VL comprises an amino acid sequence selected from any one of SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:28.

[0061] In one embodiment, the VH comprises or is selected from the group consisting of an amino acid sequence shown in any one of SEQ ID NOs: 29-33, an amino acid sequence shown in any one of SEQ ID NOs: 34-37, or an amino acid sequence shown in any one of SEQ ID NOs: 38-42;

[0062] In one embodiment, the VL comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 43-47, or the amino acid sequence shown in any one of SEQ ID NOs: 48-51, or the amino acid sequence shown in any one of SEQ ID NOs: 52-56.

[0063] In one embodiment, the VH comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 29-33, and the VL comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 43-47;

[0064] In one embodiment, the VH comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 34-37, and the VL comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 48-51.

[0065] In one embodiment, the VH comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 38-42, or the VL comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 52-56.

[0066] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 23, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 24.

[0067] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 26.

[0068] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 28.

[0069] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 43.

[0070] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 44.

[0071] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:45.

[0072] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:32, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:46.

[0073] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO: 47.

[0074] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is as shown in SEQ ID NO: 48 or 49.

[0075] In one embodiment of the isolated antibody or its antigen-binding portion described in the present invention, the amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:35, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:50.

[0076] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO:36, and the VL comprises the amino acid sequence shown in SEQ ID NO:51; or,

[0077] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO:37, and the VL comprises the amino acid sequence shown in SEQ ID NO:48; or,

[0078] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52; or,

[0079] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO:39, and the VL comprises the amino acid sequence shown in SEQ ID NO:53; or,

[0080] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO:40, and the VL comprises the amino acid sequence shown in SEQ ID NO:54; or,

[0081] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO:41, and the VL comprises the amino acid sequence shown in SEQ ID NO:55; or,

[0082] In one embodiment of the isolated antibody or antigen-binding portion thereof of the present invention, the VH comprises the amino acid sequence shown in SEQ ID NO:42, and the VL comprises the amino acid sequence shown in SEQ ID NO:56.

[0083] In some embodiments, the TIGIT-specific antigen-binding molecule of the present invention is a TIGIT antigen-binding fragment.

[0084] In one embodiment, the TIGIT binding fragment is Fab.

[0085] In one embodiment, the TIGIT binding fragment Fab',

[0086] In one embodiment, the TIGIT binding fragment F(ab)2,

[0087] In one embodiment, the TIGIT binding fragment Fv fragment,

[0088] In one embodiment, the TIGIT binding fragment F(ab')2,

[0089] In one embodiment, the TIGIT binding fragment scFv,

[0090] In one embodiment, the TIGIT binding fragment di-scFv,

[0091] In one embodiment, the TIGIT binding fragment VHH,

[0092] In one embodiment, the TIGIT binding fragment dAb.

[0093] In a fourth aspect, the present invention provides a full-length antibody that specifically binds to TIGIT, comprising a heavy chain (HC) and a light chain (LC). In one embodiment, the antibody that specifically binds to TIGIT is a full-length antibody, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain (LC) comprises a light chain variable region (VL) and a light chain constant region (CL), wherein the full-length antibody comprises one or two of the following HC and LC:

[0094] (1) a heavy chain HC, the HC comprising an amino acid sequence as shown in any one of SEQ ID NOs: 57-73, or an amino acid sequence having at least 85% homology with the amino acid sequence as shown in any one of SEQ ID NOs: 57-73, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 57-73;

[0095] (2) A light chain LC, wherein the LC comprises an amino acid sequence as shown in any one of SEQ ID NOs: 74-90, or an amino acid sequence having at least 85% homology with the amino acid sequence as shown in any one of SEQ ID NOs: 74-90, or an amino acid sequence having a sequence identity of not less than 85% with the amino acid sequence as shown in any one of SEQ ID NOs: 74-90.

[0096] In one embodiment, in the TIGIT antibody of the full-length antibody, the amino acid sequence combination of the HC and LC has at least 85% sequence identity with the amino acid sequence combination shown in any of the following groups:

[0097] a) HC: SEQ ID NO: 57 and LC: SEQ ID NO: 74;

[0098] b) HC: SEQ ID NO: 58 and LC: SEQ ID NO: 75;

[0099] c) HC: SEQ ID NO: 59 and LC: SEQ ID NO: 76;

[0100] d) HC: SEQ ID NO: 60 and LC: SEQ ID NO: 77;

[0101] e) HC: SEQ ID NO: 61 and LC: SEQ ID NO: 78;

[0102] f) HC: SEQ ID NO: 62 and LC: SEQ ID NO: 79;

[0103] g) HC: SEQ ID NO: 63 and LC: SEQ ID NO: 80;

[0104] h) HC: SEQ ID NO: 64 and LC: SEQ ID NO: 81;

[0105] i) HC: SEQ ID NO: 64 and LC: SEQ ID NO: 82;

[0106] j) HC: SEQ ID NO: 65 and LC: SEQ ID NO: 83;

[0107] k) HC: SEQ ID NO: 66 and LC: SEQ ID NO: 84;

[0108] l) HC: SEQ ID NO: 67 and LC: SEQ ID NO: 81;

[0109] o) HC: SEQ ID NO: 69 and LC: SEQ ID NO: 86;

[0110] p) HC: SEQ ID NO:70 and LC: SEQ ID NO:87;

[0111] q) HC: SEQ ID NO:71 and LC: SEQ ID NO:88;

[0112] r) HC: SEQ ID NO: 72 and LC: SEQ ID NO: 89; and

[0113] s) HC: SEQ ID NO:73 and LC: SEQ ID NO:90.

[0114] In one embodiment, the antibody that specifically binds to TIGIT further comprises a human or mouse heavy chain constant region (CH).

[0115] In one embodiment, the heavy chain constant region (CH) is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0116] In one embodiment, the CH is a human IgG4 heavy chain constant region.

[0117] In one embodiment, the CH further comprises CH1, CH2, CH3 and CL domains.

[0118] In one embodiment, the antibody that specifically binds to TIGIT further comprises a human or mouse light chain constant region (CL).

[0119] In one embodiment, the CL is a kappa or lambda chain.

[0120] In one embodiment, the CL sequence is selected from human kappa or lambda chain.

[0121] In one embodiment, the CL sequence is human κ or κ chain.

[0122] In one embodiment, the TIGIT binding antibody is a monoclonal antibody.

[0123] In one embodiment, the TIGIT binding antibody is a monoclonal antibody single chain antibody.

[0124] In one embodiment, the TIGIT binding antibody is a chimeric antibody.

[0125] In one embodiment, the TIGIT binding antibody is a humanized antibody.

[0126] In one embodiment, the anti-TIGIT antibody of the present invention is a fully human antibody.

[0127] In one embodiment, the antibody that specifically binds to TIGIT is capable of binding to both human and cynomolgus monkey TIGIT.

[0128] In a more specific embodiment, the anti-TIGIT antibodies of the present invention are capable of blocking the interaction between human or cynomolgus monkey TIGIT and the corresponding human or cynomolgus monkey CD155 / PVR receptor.

[0129] In another embodiment, the antibody is less than 5x10 -9 M or less, preferably less than 5x10 -10 M or less, even more preferably less than 5x10 -11 KD below M binds to human TIGIT.

[0130] In a fifth aspect, the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, which cross-competes with the antibody according to the present invention described herein for binding to TIGIT.

[0131] In the sixth aspect, the isolated anti-TIGIT monoclonal antibody or its antigen-binding portion described in the present invention (a) binds to human TIGIT; (b) binds to monkey TIGIT; (c) does not bind to mouse TIGIT; (d) blocks TIGIT-PVR interaction; (e) promotes T cell activation; (f) triggers ADCC of immune cells against TIGIT-positive cells; (g) has an anti-tumor effect in vivo; and / or (h) has a synergistic anti-tumor effect with PD L1 antibody.

[0132] In a seventh aspect, the present invention provides a polypeptide comprising the isolated antibody or antigen-binding fragment thereof according to any of the preceding aspects.

[0133] In an eighth aspect, the present invention provides an immunoconjugate comprising the isolated antigen-binding protein or polypeptide according to any one of the preceding claims.

[0134] In a ninth aspect, the present invention provides an isolated nucleic acid encoding a polypeptide, or an antibody light chain or heavy chain, or a variable region sequence of an anti-TIGIT antibody or antigen-binding fragment thereof as described herein,

[0135] In one embodiment, the isolated nucleic acid encoding the heavy chain variable region comprises a nucleotide sequence selected from any one of SEQ ID NOs: 186-202, or comprises a nucleotide sequence selected from any one of SEQ ID NOs: 186-202 having a sequence homology of not less than 85%.

[0136] In one embodiment, the isolated nucleic acid encoding the light chain variable region is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 203-219, or comprises a nucleotide sequence selected from the nucleotide sequence shown in any one of SEQ ID NOs: 203-219 having a sequence homology of not less than 85%.

[0137] In a tenth aspect, the present invention provides a vector suitable for expressing one or more of the above-mentioned isolated nucleic acids.

[0138] In the eleventh aspect, the present invention provides a host cell comprising the above-mentioned isolated nucleic acid molecule, the above-mentioned vector, and the above-mentioned polypeptide, which is suitable for expressing the nucleic acid and delivering the mature, correctly folded polypeptide of the anti-TIGIT antibody or its antigen-binding fragment as described herein, or the antibody light chain or heavy chain, or the variable region sequence.

[0139] In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

[0140] In a specific embodiment, the eukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary (CHO).

[0141] In a more specific embodiment, the CHO cell is CHO-K1.

[0142] In the twelfth aspect, the present invention provides a method for producing the above-mentioned anti-TIGIT antibody or its antigen-binding fragment, comprising: (1) preparing a murine anti-TIGIT antibody by the hybridoma method; (2) culturing a host cell containing a nucleic acid encoding the TIGIT antibody or antigen-binding fragment described in any of the preceding aspects in a form suitable for expression under conditions suitable for producing the antibody or fragment, and recovering the antibody or fragment.

[0143] In the thirteenth aspect, the present invention provides a pharmaceutical composition comprising the antibody specifically binding to TIGIT or its antigen-binding fragment of the aforementioned aspect, the aforementioned immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and a combination of at least one pharmaceutically acceptable adjuvant and / or excipient.

[0144] In one embodiment, the present invention provides a pharmaceutical composition comprising the isolated TIGIT antigen binding protein described in any one of the above and an immune checkpoint inhibitor.

[0145] In one embodiment, the immune checkpoint inhibitor comprises a substance that inhibits PD-L1 interaction, or alternatively, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

[0146] In a fourteenth aspect, the present invention provides the antibody specifically binding to TIGIT or its antigen-binding fragment, the above-mentioned immunoconjugate, the isolated nucleic acid molecule, the vector, and the cell as described in the previous aspects, which are used to prevent, alleviate and / or treat tumors, wherein the tumor includes a solid tumor, or the tumor includes a tumor associated with the expression of a group of proteins selected from the group consisting of TIGIT, CD155 and CD112.

[0147] In the fifteenth aspect, the present invention provides the aforementioned antibody that specifically binds to TIGIT or its antigen-binding fragment, the aforementioned immunoconjugate, the aforementioned isolated nucleic acid molecule, the aforementioned vector, the aforementioned cell, in combination with at least one pharmaceutically acceptable carrier; a pharmaceutical composition comprising the isolated TIGIT antigen-binding protein described in any of the above and an immune checkpoint inhibitor, and its use in the preparation of disease prevention and / or treatment drugs.

[0148] In one embodiment, the disease is cancer.

[0149] In one embodiment, the pharmaceutical composition comprises any one or a combination of at least two of the anti-TIGIT monoclonal antibody described in the first aspect, the humanized anti-TIGIT monoclonal antibody described in the second aspect, the humanized anti-TIGIT monoclonal antibody variant described in the third aspect, or the anti-TIGIT / PD-1 bispecific antibody described in the fourth aspect.

[0150] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

[0151] In a sixteenth aspect, the present invention provides a kit comprising a pharmaceutical composition disclosed herein, an anti-cancer agent, and a package insert comprising instructions for using the anti-cancer agent in combination with an anti-TIGIT antibody to treat T cell dysfunction diseases and / or cancer in an individual.

[0152] In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head and neck cancer, gastric cancer and pancreatic cancer.

[0153] In the seventeenth aspect, the present invention provides antibodies and variants thereof that specifically bind to TIGIT according to the aforementioned aspects, compositions comprising the same, drug conjugates comprising the same, and uses of the nucleic acids encoding the same in the preparation of drugs that enhance T cell function.

[0154] In one embodiment, the method for testing the drug comprises administering an effective amount of any of the above-mentioned anti-TIGIT antibodies or compositions.

[0155] In the eighteenth aspect, the present invention provides the use of antibodies and variants thereof that specifically bind to TIGIT according to the aforementioned aspects, compositions comprising the same, drug conjugates comprising the same, and nucleic acids encoding the same in the preparation of a medicament for increasing, enhancing or stimulating the immune response or function of an individual suffering from cancer, wherein the method for administering the medicament comprises administering to the individual an effective amount of an antibody that specifically binds to TIGIT and an anti-cancer agent and / or anti-cancer therapy.

[0156] In the nineteenth aspect, the present invention provides the use of antibodies and variants thereof that specifically bind to TIGIT of the aforementioned aspects, compositions comprising the same, drug conjugates comprising the same, and nucleic acids encoding the same in the preparation of drugs for treating or delaying the progression of tumor immunity or cancer, or reducing or inhibiting the recurrence of cancer in individuals, wherein the method of administering the drug comprises administering an effective amount of an antibody that specifically binds to TIGIT and an anticancer agent and / or anticancer therapy to the individual.

[0157] In certain embodiments, the method comprises administering to the individual an effective amount of an antibody that specifically binds TIGIT and / or an anti-cancer agent and / or an anti-cancer therapy.

[0158] In certain embodiments, the anti-cancer therapy is selected from the group consisting of radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, adjuvant therapy, neoadjuvant therapy, hormone therapy, angiogenesis inhibition, palliative treatment, and combinations thereof.

[0159] In a twentieth aspect, the present invention relates to the use of the antibody or composition specifically binding to TIGIT disclosed herein in the preparation of a drug for cancer treatment.

[0160] The cancer treatment comprises administering to a subject in need thereof an effective amount of an anti-TIGIT antibody that induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0161] Preferably, the disease includes any one of cancer, viral infectious disease or immune system disease, or a combination of at least two thereof.

[0162] Preferably, the cancer comprises any one of lung cancer, kidney cancer, melanoma, breast cancer, liver cancer, head and neck cancer, skin cancer, squamous cell carcinoma, ovarian cancer, bone cancer, colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, prostate cancer, Hodgkin's lymphoma, follicular lymphoma, chronic leukemia or acute leukemia, or a combination of at least two thereof.

[0163] Preferably, the viral infectious disease includes any one of HIV viral infectious disease, hepatitis viral infectious disease, herpes viral infectious disease or influenza viral infectious disease, or a combination of at least two of them. Preferably, the immune system disease includes any one of lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, scleroderma, polyarteritis nodosa or Wegener's granulomatosis, or a combination of at least two of them.

[0164] Beneficial effects:

[0165] Compared with the prior art, the present invention has the following beneficial effects:

[0166] (1) The mouse anti-human TIGIT monoclonal antibody of the present invention has significant specific binding ability to human TIGIT protein and also has binding activity to TIGIT antigen expressed by cells, and can effectively block the binding of TIGIT to its ligand CD155 at the cellular level;

[0167] (2) Compared with the mouse anti-TIGIT monoclonal antibody, the humanized anti-human TIGIT monoclonal antibody of the present invention has no significant decrease in binding activity to the human TIGIT antigen and has binding activity to the TIGIT antigen on the surface of CHO K1 cells;

[0168] (3) The seven humanized anti-TIGIT monoclonal antibody variants of the present invention have high affinity to the TIGIT antigen, can block the binding of TIGIT to CD155, and have the potential to reactivate T cells and relieve immunosuppression;

[0169] (4) The anti-TIGIT / PD-1 bispecific antibody of the present invention has the ability to bind to both TIGIT and PD-1, and exerts an anti-tumor effect by blocking the interaction between CD155-TIGIT and PD-L1-PD-1. It has broad application prospects in the preparation of tumor prevention and / or treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figure 1 ELISA analysis of TIGIT protein binding activity of mouse monoclonal antibody

[0171] Figure 2 ELISA analysis of the blocking activity of TIGIT mouse monoclonal antibody in blocking the binding of TIGIT to CD155

[0172] Figure 3 FACS analysis of the binding activity of mouse monoclonal antibody against TIGIT and TIGIT on the surface of CHO-K1-TIGIT cells

[0173] Figure 4 FACS analysis of the blocking activity of TIGIT mouse monoclonal antibody in blocking the binding of CHO-K1-TIGIT to CD155 at the cellular level

[0174] Figure 5 ELISA analysis of TIGIT protein binding activity of TIGIT chimeric antibodies BT-25H8-CHI, BT-7F4-CHI, and BT-6A10-CHI

[0175] Figure 6 ELISA analysis of the blocking activity of TIGIT chimeric antibodies BT-25H8-CHI, BT-7F4-CHI, and BT-6A10-CHI in blocking the binding of TIGIT to CD155

[0176] Figure 7FACS binding activity analysis of TIGIT chimeric antibodies BT-25H8-CHI, BT-7F4-CHI, and BT-6A10-CHI binding to TIGIT on the surface of CHO-K1-TIGIT cells

[0177] Figure 8 FACS blocking activity analysis of the blocking activity of TIGIT chimeric antibodies BT-25H8-CHI, BT-7F4-CHI, and BT-6A10-CHI in blocking the binding of CHO-K1-CD155TIGIT to CD155

[0178] Fig. 9 , affinity analysis of antibody BT-25H8-CHI and antigen TIGIT-HIS was detected based on surface plasmon resonance (SPR) technology.

[0179] Fig.10 , affinity analysis of antibody BT-7F4-CHI and antigen TIGIT-HIS was detected based on surface plasmon resonance (SPR) technology.

[0180] Fig.11 , affinity analysis of antibody BT-6A10-CHI and antigen TIGIT-HIS was detected based on surface plasmon resonance (SPR) technology. Fig.12 ELISA analysis of TIGIT protein binding activity of humanized TIGIT antibodies BT-25H8-12, BT-25H8-11, BT-25H8-32, BT-7F4-42, BT-7F4-41, BT-7F4-44, BT-7F4-43 and Tiragolumab

[0181] Fig.13 ELISA analysis of the blocking activity of TIGIT humanized antibodies BT-25H8-12, BT-25H8-11, BT-25H8-32, BT-7F4-42, BT-7F4-41, BT-7F4-44, BT-7F4-43 and Tiragolumab in blocking the binding of TIGIT to CD155

[0182] Fig.14 FACS binding activity analysis of TIGIT humanized antibodies BT-25H8-12, BT-25H8-11, BT-25H8-32, BT-7F4-42, BT-7F4-41, BT-7F4-44, BT-7F4-43 and TiragolumabI binding to TIGIT on the surface of CHO-K1-TIGIT cells

[0183] Fig.15FACS blocking activity analysis of the blocking activity of humanized TIGIT antibodies BT-25H8-12, BT-25H8-11, BT-25H8-32, BT-7F4-42, BT-7F4-41, BT-7F4-44, BT-7F4-43 and Tiragolumab in blocking the binding of CHO-K1-CD155TIGIT to CD155

[0184] Fig.16 , Analysis of the binding kinetics and affinity between TIGIT chimeric antibody BT-6A10-31 and recombinant human TIGIT-His based on biofilm interferometry

[0185] Fig.17 , Analysis of the binding kinetics and affinity between TIGIT chimeric antibody BT-25H8-41 and recombinant human TIGIT-His based on biofilm interferometry

[0186] Fig.18 , Analysis of the binding kinetics and affinity between TIGIT chimeric antibody BT-6A10-33 and recombinant human TIGIT-His based on biofilm interferometry

[0187] Fig.19 , Analysis of the binding kinetics and affinity between TIGIT chimeric antibody BT-6A10-32 and recombinant human TIGIT-His based on biofilm interferometry

[0188] Fig. 20 ELISA binding activity analysis of cross-binding of TIGIT chimeric antibodies 25H8-CHI, 7F4-CHI and 6A10-CHI with cynomolgus monkey TIGIT protein.

[0189] Fig.21 ELISA binding activity analysis of cross-binding of TIGIT chimeric antibodies 25H8-CHI, 7F4-CHI and 6A10-CHI with mouse TIGIT protein.

[0190] Fig. 22 ELISA binding activity analysis of cross-binding of TIGIT humanized antibodies BT-6A10-31, BT-6A10-32, BT6A10-34, BT-6A10-33, BT-7F4-12, BT-7F4-14 and BT-25H8041 with mouse TIGIT protein

[0191] Fig.23ELISA binding activity analysis of cross-binding of TIGIT humanized antibodies BT-6A10-31, BT-6A10-32, BT6A10-34, BT-6A10-33, BT-7F4-12, BT-7F4-14 and BT-25H8041 with mouse TIGIT protein

[0192] Fig.24 Schematic diagram of the mammalian expression vector PHR used in Example 1

[0193] Fig.25 Schematic diagram of TIGIT / CD155 binding initiating a major inhibitory signal through an ITT-like motif

[0194] definition

[0195] Antibody related definitions

[0196] The term "antibody" includes monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies, diabodies) and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2 and Fv). The term "immunoglobulin" (Ig) can be used interchangeably with "antibody" herein.

[0197] Based on the amino acid sequence of its constant domain, the L chain of any vertebrate species can be classified into one of two clearly distinct types, called κ (kappa) and λ (lambda). Depending on the amino acid sequence of its heavy chain constant domain (CH), immunoglobulins can be classified into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes are further divided into subclasses, for example, humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0198] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen binding site.

[0199] The term "variable" refers to the fact that certain fragments of the variable domain differ greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of the antibody for its antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three fragments called hypervariable regions (HVRs) in the light and heavy chain variable domains, which complement the antigen epitope structure, so the hypervariable region is also called the complementarity-determining region (CDR) of the antibody molecule. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, which mainly adopt a β-stack configuration, connected by three HVRs, forming a loop connecting the β-stack structure, and in some cases forming part of the β-stack structure. The HVRs in each chain are held together by the FR regions and, together with the HVRs in the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

[0200] The term "hypervariable region", "HVR" or "HV", when used herein, refers to a region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. In general, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity among the six HVRs, and H3 in particular is believed to play a unique role in conferring good specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (Lo ed., Human Press, Totowa, NJ, 2003). ImMunGeneTics (IMGT) unique Lefranc numbering (IMGT numbering) (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)) takes into account sequence conservation, structural data from X-ray diffraction studies, and features of hypervariable loops to define FRs and HVRs. Kabat complementarity determining regions (CDRs) are also commonly used based on sequence variability (Kabat et al., Immunology Focuses on Protein Sequences, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). In contrast, Chothia refers to the position of structural loops (Chothia and Lesk, / . Mol. Biol. 196: 901-917 (1987)). AbMHVR represents a compromise between Kabat HVR and Chothia structural loops and is used in Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of existing complex crystal structures. HVRs may include the following "extended HVRs": 24-40 (L1), 56-69 (L2), and 105-117 (L3) in VL, 24-40 (H1), 55-74 (H2), and 105-117 (H3) in VH. For these definitions, variable domain residues are numbered according to Lefranc et al., supra.

[0201] "Variable domain residue numbering using the IMGT definition" or "amino acid position numbering in IMGT" and variations thereof refer to the numbering system used for antibody variable domains in the above-mentioned reference by Lefranc et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or HVR of the variable domain. For example, the heavy chain variable domain may include residues inserted after heavy chain HVR residue 111 or before heavy chain HVR 112 (e.g., residues 111.1 and 112.1 according to Lefranc, etc.). For a given antibody, the IMGT numbering of the residues can be determined by aligning the sequence with the "standard" IMGT numbering in a homologous region of the antibody sequence.

[0202] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0203] A "human consensus framework" or "acceptor human framework" is a framework that represents the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Typically, the selected human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. In general, the subgroup of sequences is a subgroup such as Kabat et al. in "Protein Sequences of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, for VL, the subgroup may be a kappa I, kappa II, kappa III, or kappa IV subgroup in the above-mentioned literature of Kabat et al. In addition, for VH, the subgroup may be a subgroup I, a subgroup II, or a subgroup III in the above-mentioned literature of Kabat et al. Alternatively, the human consensus framework may be derived from the above-mentioned content of specific residues therein, such as when the human framework residues are selected based on their homology to the donor framework, by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may comprise pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0204] The "VH subgroup I consensus framework" comprises the consensus sequence obtained from the amino acid sequences of variable heavy subgroup I in the above-mentioned document by Kabat et al.

[0205] The "VH kappa I consensus framework" comprises the consensus sequence obtained from the amino acid sequences of variable light kappa subgroup I in Kabat et al., supra.

[0206] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residues at the Cys226 or Pro230 positions to its carboxyl terminus. Unless otherwise specified herein, the residue numbering in the constant region of an immunoglobulin heavy chain is the EU index, as described in the literature of Kabat et al. "The EU index in Kabat" refers to the residue numbering of human IgG1 EU antibodies. The C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody, or by recombinant engineering encoding an antibody heavy chain nucleic acid.

[0207] Therefore, the composition of the whole antibody can include an antibody population with all K447 residues removed, an antibody population with no K447 residue removed, and an antibody population with a mixture of antibodies containing and not containing the K447 residue. Suitable native sequence Fc regions for antibodies of the present invention include human IgG1, IgG2, IgG3, and IgG4. "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. In addition, the preferred FcR is an FcR that binds to IgG antibodies (gamma receptors) and includes FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, mainly different in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, see M. Daeron, Annu. Rev. Immunol. 15: 203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are included in the term "FcR" herein.

[0208] The "Fc fragment" comprises the carboxyl terminal portions of two H chains held together by disulfide bonds. The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcR) on certain types of cells.

[0209] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain and a light chain variable region domain in tight non-covalent association. The folding of these two domains produces six hypervariable loops (three loops each in the H chain and L chain), which provide amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigens.

[0210] "Amino acid modification" at a specified position (e.g., an Fc region) refers to substitution or deletion of a specified residue, or insertion of at least one amino acid residue adjacent to a specified residue. Insertion "adjacent" a specified residue means insertion within one to two residues thereof. Insertion may be at the N-terminus or C-terminus of a specified residue. Preferred amino acid modifications herein are substitutions.

[0211] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or a radiolabel.

[0212] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antibody fragment. Specifically, an intact antibody includes an antibody having heavy and light chains, including an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof.

[0213] In some cases, an intact antibody may possess one or more effector functions.

[0214] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. The Fab fragment consists of an intact L chain as well as the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent in terms of antigen binding, i.e., has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F

[0215] (ab')2 fragments, which roughly correspond to two disulfide-linked Fab fragments with different antigen binding activities, and are still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that there are several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry free sulfhydryl groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0216] "Single-chain Fv", also referred to as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide also comprises a polypeptide connector between the VH and VL domains, which enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0217] The "functional fragment" of the antibody of the present invention comprises a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody, or the Fc region of an antibody that retains FcR binding ability or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed by antibody fragments.

[0218] The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment (see above) with a short linker (about 5-10 residues) between the VH and VL domains to achieve interchain rather than intrachain pairing of the V domains, thereby generating a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of these antibodies, so long as they exhibit the desired biological activity.

[0219] "Humanized" forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody), wherein residues from the HVR (defined below) of the receptor are replaced by residues from the HVR of a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates with desired specificity, affinity, and / or ability. In some cases, the skeleton ("FR") residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in residues not found in a receptor antibody or a donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one (usually two) variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are the hypervariable loops of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions to improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is usually no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986) Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., U.S. Patent Nos. 6,982,321 and 7,087,409.

[0220] "Human antibodies" are antibodies with amino acid sequences corresponding to the amino acid sequences of antibodies produced by humans, and / or antibodies prepared using any of the techniques for preparing human antibodies disclosed herein. The definition of human antibodies explicitly excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries, or by administering antigens to transgenic animals, which are genetically modified to produce partial or complete human antibodies in response to antigen introduction, but whose endogenous loci have been disabled. Regarding human antibodies produced by human B cell hybridoma technology, see also, for example, Li et al., Proc. Natl. Acad. Set USA, 103: 3557-3562 (2006).

[0221] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Different from polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the property of the antibody is obtained from a substantially homogeneous antibody population and should not be construed as requiring the production of antibodies by any particular method.For example, the monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas, 1996). Hybridomas)》563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Sidhu et al. J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004)), and techniques for producing human or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004)). 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar Intern. Rev. Immunol. 13:65-93 (1995)).

[0222] An "affinity matured" antibody refers to an antibody that has one or more changes in one or more HVRs that result in an improved affinity of the antibody for the antigen compared to a parent antibody that does not have these changes. In one embodiment, the affinity matured antibody has a nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by processes known in the art. For example, Marks et al. describe affinity maturation by shuffling of the VH and VL domains in Bio / Technology 10:779-783 (1992). Random mutagenesis of HVR and / or framework residues is described, for example, in Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0223] As used herein, the term "specific binding" or "specific for" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which is deterministic for the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to the target with greater affinity, avidity, ease, and / or for a longer period of time than it binds to other targets.

[0224] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the expression or biological activity of the antigen. For example, an antibody or antigen-binding fragment thereof that specifically binds to TIGIT of the present invention can inhibit TIGIT expression, block the interaction between TIGIT and PVR, block the interaction between TIGIT and PVRL2, block the interaction between TIGIT and PVRL3, inhibit and / or block intracellular signaling mediated by TIGIT binding to PVR, inhibit and / or block intracellular signaling mediated by TIGIT binding to PVRL2, and / or inhibit and / or block intracellular signaling mediated by TIGIT binding to PVRL3.

[0225] An "agonist" or activating antibody is an antibody that enhances or initiates signaling through the antigen to which it binds. In some embodiments, an agonist antibody causes or activates signaling in the absence of a natural ligand.

[0226] The terms "cross-competition" and "cross-blocking" are used interchangeably herein and refer to the ability of an antibody or fragment thereof to interfere with binding directly or indirectly through allosteric modulation of the antibody of the invention to its target antigen. The extent to which an antibody or fragment thereof can interfere with the binding of another antibody to a target, and therefore whether it can be called cross-blocking or cross-competing according to the invention, can be determined using a competitive binding assay. A particularly suitable quantitative cross-competition assay uses a FACS-based or AlphaScreen-based method to measure competition between a labeled (e.g., His-tagged, biotin-tagged or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof in terms of binding to a target. In general, a cross-competing antibody or fragment thereof is, for example, an antibody or fragment thereof that will bind to a target in a cross-competition assay, such that during the assay and in the presence of a second antibody or fragment thereof, the recorded displacement of an immunoglobulin single variable domain or polypeptide according to the invention (e.g., in a FACS-based competition assay) is up to 100% of the maximum theoretical displacement of a potential cross-blocking antibody or fragment thereof to be tested in a certain number of presences (e.g., the displacement of a cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked). Preferably, the cross-competing antibody or fragment thereof has a recorded shift between 10% and 100%, more preferably between 50% and 100%.

[0227] "Bispecific antibodies" are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificity for at least two different antigens. Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, wherein the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).

[0228] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity," "binding to," or "binding to" refers to the intrinsic binding affinity that reflects a 1-to-1 interaction between members of a binding pair (e.g., an antibody Fab fragment and an antigen). The affinity of a molecule X and its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including methods described herein. Low-affinity antibodies generally bind slowly to antigens and are easy to dissociate, while high-affinity antibodies generally bind faster to antigens and remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity (i.e., binding strength) are described below.

[0229] The "KD" or "KD value" according to the present invention can be measured by using the Fab version of the antibody and the antigen molecule using the Bio layer interferometry (BLI) technique, or by using surface plasmon resonance analysis using a BIACORE instrument (BIAcore, Inc., Piscataway, NJ).

[0230] Immune system related definitions

[0231] "Immunogenicity" refers to the ability of a substance to stimulate an immune response. Tumors are immunogenic, and enhancing the immunogenicity of tumors helps to eliminate tumor cells through immune response. Examples of enhancing tumor immunogenicity include, but are not limited to, treatment with immunomodulatory receptor inhibitors.

[0232] "Enhancing T cell function" means inducing, causing or stimulating T cells to have a continuous or amplified biological function, or renewing or reactivating exhausted or inactivated T cells. Examples of enhancing T cell function include: compared with the level before intervention, CD8+T cells have increased secretion of gamma interferon, increased proliferation, and increased antigenic reactivity (e.g., virus, pathogen or tumor clearance). In one embodiment, the enhancement level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The method of measuring this enhancement is known to those of ordinary skill in the art.

[0233] A "T cell dysfunctional disease" is a disease or condition of T cells characterized by reduced responsiveness to antigenic stimulation (e.g., to a tumor expressing an immunogen). For example, a T cell dysfunctional disease may be characterized by T cell anergy or a decreased ability to secrete cytokines, proliferate, or perform cytolytic activities. Reduced responsiveness may result in ineffective control of tumors expressing immunogens. T cell dysfunctional diseases characterized by T cell dysfunction include tumor immunity and cancer.

[0234] In the context of immune dysfunction, the term "dysfunction" refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes common factors that antigen recognition may occur but the subsequent failure and / or inability of the immune response to control infection or tumor growth is ineffective.

[0235] As used herein, the term "dysfunctional" also includes refractory or unresponsiveness to antigen recognition, specifically, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0236] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when this escape is attenuated and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0237] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FCRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells carrying antigens and then use cytotoxins to kill the target cells. Antibodies "arm" cytotoxic cells and kill target cells through this mechanism. The primary cells used to mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 can be performed. Useful effector cells for such analysis include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0238] "Effector cells" are leukocytes that express one or more FCRs and perform effector functions. On the one hand, effector cells express at least FcγRIII and perform ADCC effector functions. Human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be separated from natural sources (e.g., blood). Effector cells are typically lymphocytes associated with the effector phase, and their function is to produce cytokines (helper T cells), kill cells that infect pathogens (cytotoxic T cells), or secrete antibodies (differentiated B cells).

[0239] "Autoimmune disease" is a disease or disorder caused by or arising from an individual's own tissues or organs or their co-isolation or manifestation. An autoimmune disease can be an organ-specific disease (i.e., the immune response is specific to one organ system, such as the endocrine system, hematopoietic system, skin, cardiopulmonary system, gastrointestinal and liver system, kidney system, thyroid, ear, neuromuscular system, central nervous system, etc.), or a systemic disease that can affect multiple organ systems (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis, etc.).

[0240] Cancer-related definitions

[0241] As used herein, "tumor" refers to all malignant or benign tumor cell growth and proliferation, as well as all precancerous and cancerous cells and tissues. The terms "cancer", "carcinogenesis", "cell proliferative disease", "proliferative disease" and "tumor" are not mutually exclusive in this article.

[0242] As used herein, "cancer" and "cancerous change" refer to or describe the physiological condition in mammals that is typically characterized by disordered cell growth. The definition includes benign and malignant tumors as well as dormant tumors or micrometastases.

[0243] As used herein, "metastasis" means the spread of cancer from its primary site to other parts of the body. Cancer cells can break away from the primary tumor, infiltrate into lymphatic and blood vessels, circulate in the blood, and grow (metastasis) in distant lesions in normal tissues in other parts of the body. Metastasis can be local or distant. Metastasis is a continuous process that depends on tumor cells that break away from the primary tumor, move in the bloodstream, and stop at a distant site. At the new site, these cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within tumor cells regulate this behavior, and the interaction between tumor cells and host cells in distant sites is also important.

[0244] As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and / or cancer progression include, but are not limited to, cancer metastasis.

[0245] Definitions related to formulation and drug delivery

[0246] The term "pharmaceutical preparation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered. Such preparations are sterile.

[0247] A "sterile" preparation is sterile, or free from all living microorganisms and their spores.

[0248] The term "about" as used herein refers to the normal error range for the respective value which is readily known to one skilled in the art.

[0249] The phrase "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising the formulation and / or the individual to be treated therewith.

[0250] A "stable" formulation is one in which the protein substantially maintains its physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in the following literature: "Peptide and Protein Drug Delivery", 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991). Stability can be measured at a selected temperature and a selected time period. For rapid screening, the formulation can be stored at 40°C for 2 weeks to 1 month, during which stability is measured. If the formulation will be stored at 2-8°C, the formulation should generally be stable at 30°C or 40°C for at least 1 month, and / or stable at 2-8°C for at least 2 years. If the formulation will be stored at 30°C, the formulation should generally be stable at 30°C for at least 2 years and / or stable at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one in which less than about 10%, and preferably less than about 5%, of the protein is present in the formulation as aggregates.

[0251] A "reconstituted" formulation is one that is prepared by dissolving a lyophilized protein or antibody formulation in a diluent so that the protein is well dispersed. The reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient to be treated with the protein of interest, and in certain embodiments of the invention, may be a formulation suitable for parenteral or intravenous administration.

[0252] An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of about 250 to 350 mOsm. The term "hypertonic" describes a formulation that has an osmotic pressure higher than human blood. Accordingly, the term "hypertonic" is used to describe a formulation that has an osmotic pressure higher than human blood. For example, isotonicity can be measured using a vapor pressure or ice-type osmometer.

[0253] "Pharmaceutically acceptable" buffers and salts include those derived from both acid and base addition salts of the above-mentioned acids and bases. Specific buffers and / or salts include histidine, succinate, and acetate.

[0254] As used herein, "pharmaceutically acceptable carrier" refers to an excipient that is nontoxic to cells or individuals exposed thereto at the dosages and concentrations employed. Typically, a pharmaceutically acceptable carrier is a pH buffered aqueous solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, polyethylene glycol (PEG), and Pluronics.

[0255] “Package insert” means instructions customarily included in commercial packages of drugs that contain information about the indications, usage, dosage, administration, contraindications, other drugs combined with the packaged product, and / or warnings concerning the use of such drug.

[0256] Other definitions

[0257] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of an individual or cell being treated during a clinical pathological process (e.g., cancer or tumor immunity). Ideal therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing (or destroying) the proliferation of cancerous cells, reducing symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dose of other drugs required to treat the disease, delaying disease progression, and / or prolonging the survival of an individual, the individual is successfully "treated".

[0258] As used herein, "delaying disease progression" means postponing, hindering, slowing down, blocking, stabilizing and / or delaying the development of a disease (e.g., cancer or tumor immunity). Depending on the history of the disease and / or the individual being treated, this delay can have various time lengths. It will be apparent to those skilled in the art that sufficient or significant delay can actually encompass prevention, because then the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

[0259] "Effective amount" is at least the minimum concentration required to cause a measurable improvement or prevention of a disease. The effective amount herein may vary according to factors such as the patient's disease state, age, sex and weight, and the ability of the antibody to elicit a desired response in an individual. An effective amount is also an amount in which any toxic or harmful effects of a treatment are exceeded by the beneficial effects of the treatment. For preventive use, beneficial or desired results include the results of eliminating or reducing risk, reducing severity, or delaying the onset of a disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during the course of disease development. For therapeutic use, beneficial or desired results include the following clinical results, such as reducing one or more symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dose of other drugs required to treat the disease, enhancing the effects of other drugs by, for example, targeting, delaying disease progression, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may have the following effects: reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing down or intentionally stopping to some extent) cancer cell infiltration into peripheral organs; inhibiting (i.e., slowing down and intentionally stopping to some extent) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with the disease to some extent. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly complete a preventive or therapeutic treatment. As understood in a clinical setting, an effective amount of a drug, compound or pharmaceutical composition can be achieved with or without another drug, compound or pharmaceutical composition.

[0260] Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if it can achieve or accomplish the desired result when administered in combination with one or more other agents.

[0261] As used herein, "combination" refers to administering one treatment modality in addition to another treatment modality. Thus, "combination" refers to administering one treatment modality before, during, or after another treatment modality is administered to an individual.

[0262] The terms "used in conjunction with" and "used in conjunction with" are used interchangeably herein.

[0263] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0264] References herein to "about" a value or parameter include (and describe) variations to that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X."

[0265] As used herein, the terms "individual" and "subject" are used interchangeably and refer to mammals, including but not limited to humans or non-human mammals, such as cattle, horses, dogs, sheep or cats. Preferably, the individual or subject is a human. A patient is also an individual or subject herein.

[0266] "Percent (%) amino acid sequence identity" and "homology" of peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2 or ALIGN software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0267] The "isolated" nucleic acid molecule encoding the polypeptide and antibody herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule, which is usually associated with the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with all components associated with the production environment. The form of the isolated nucleic acid molecule encoding the polypeptide and antibody herein is different from the form or environment in which it is found in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptide and antibody herein that naturally occurs in the cell.

[0268] As used herein, the phrase "substantially reduced" or "substantially different" means a sufficiently high degree of difference between two values ​​(usually one associated with a molecule and the other associated with a reference / comparator molecule) such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological property measured by the value (e.g., KD value). The difference between the two values ​​as a function of the value of the reference / comparator molecule is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%.

[0269] As used herein, the term "substantially similar" or "substantially identical" refers to a sufficiently high degree of similarity between two values ​​(e.g., one associated with an antibody of the invention and the other associated with a reference / comparison antibody) such that a person skilled in the art would consider the difference between the two values ​​to be of little or no biological and / or statistical significance in the context of a biological property measured by the value (e.g., Kd value). The difference between the two values ​​as a function of a reference / comparison value is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%.

[0270] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the natural polypeptides disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of the natural polypeptides disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. The method of identifying an agonist or antagonist of a polypeptide can include contacting the polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the polypeptide.

[0271] The terms "TIGIT antagonist" and "antagonist of TIGIT activity or TIGIT expression" are used interchangeably and refer to compounds that interfere with the normal function of TIGIT by reducing the transcription or translation of nucleic acids encoding TIGIT, or by inhibiting or blocking the activity of TIGIT polypeptides, or both. Examples of TIGIT antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragments of anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-binding peptides, and other polypeptides that specifically bind to TIGIT (including but not limited to TIGIT-binding fragments of one or more TIGIT ligands, optionally fused to one or more additional domains), such that the interaction between the TIGIT antagonist and TIGIT results in a reduction or cessation of TIGIT activity or expression. It should be understood by those of ordinary skill in the art that, in some cases, a TIGIT antagonist may antagonize one TIGIT activity without affecting another TIGIT activity. For example, an ideal TIGIT antagonist for use in certain methods herein is a TIGIT antagonist that antagonizes TIGIT activity in response to one of a PVR interaction, a PVRL3 interaction, or a PVRL2 interaction, e.g., without affecting or minimally affecting any other TIGIT interaction.

[0272] Various aspects of the present application are described in more detail below. The antibodies of the present application can specifically bind to human and monkey TIGIT, and the binding activity is comparable to or better than the prior art TIGIT antagonist antibodies such as Tiragolumab or Etigilimab. The antibodies of the present application can also block TIGIT-PVR binding or interaction, and the blocking activity is comparable to the prior art antibodies such as Tiragolumab or Etigilimab.

[0273] More importantly, the antibodies of the present application have TIGIT binding activity comparable to or stronger than prior art antibodies such as Tiragolumab or Etigilimab, stronger T cell activation ability, and in vivo anti-tumor activity comparable to or even higher than prior art antibodies such as Tiragolumab or Etigilimab.

[0274] Preferred antibodies of the present application are monoclonal antibodies. In addition, the antibody can be, for example, a murine, chimeric or humanized monoclonal antibody.

[0275] Exemplary antibodies of the present application are monoclonal antibodies whose structures and chemical properties are described below.

[0276] The present inventors creatively prepared chimeric antibodies against human TIGIT (named 25H8(464)-CHI, 7F4(465)-CHI and 6A10(467)-CHI), and humanized antibodies (named 25H8-1(H4A+L1A), 25H8-2(H4B+L2A), 25H8-3(H4C+L3A), 25H8-4(H4D+L6A), 25H8-5(H2A+L1B ), 7F4-1(H1A+L2A), 7F4-2(H1A+L4A), 7F4-3(H1B+L1A), 7F4-4(H7A+L2B), 7F4-5(H3A+L2A), 6 A10-1(H3A+L1A), 6A10-2(H3B+L2A), 6A10-3(H3C+L4A), 6A10-4(H3D+L6A) and 6A10-5(H3E+L3A).

[0277] Antigen binding activity, blocking activity and affinity and functional activity

[0278] The present inventors also surprisingly found that the chimeric antibodies 25H8(464)-CHI, 7F4(465)-CHI and 6A10(467)-CHI antibodies and the fully humanized antibodies 25H8-1 (H4A+L1A), 25H8-2 (H4B+L2A), 25H8-3 (H4C+L3A), 25H8-4 (H4D+L6A), 25H8-5 (H2A+L1B), 7F4-1 (H1A+L2A), 7F4-2 (H1A+L4A), 7F4-3 (H1B+L 1A), 7F4-4 (H7A+L2B), 7F4-5 (H3A+L2A), 6A10-1 (H3A+L1A), 6A10-2 (H3B+L2A), 6A10-3 (H3C+L4A), 6A10-4 (H3D+L6A) and 6A10-5 (H3E+L3A) have the activity of high-level specific binding to TIGIT and have extremely strong affinity; they can effectively reduce the activity of TIGIT and the inhibition of contact immune cell activation, thereby improving the tumor inhibition rate or killing rate.

[0279] In addition, the present inventors found that the anti-TIGIT antibody of the present disclosure combined with the anti-PD-1 antibody can effectively prevent and treat tumors.

[0280] The antibodies of the present invention have the potential to be used for treating and / or preventing diseases such as tumors (e.g., non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, plasma cell cancer). The following invention is provided:

[0281] In one embodiment, the present application provides an isolated murine TIGIT antibody that binds to TIGIT-hFc with an ELISIA binding EC50 of no more than 20 ng / ml, preferably no more than 15 ng / ml, more preferably no more than 12 ng / ml, more preferably no more than 10 ng / ml, more preferably no more than 8 ng / ml, further more preferably no more than 5 ng / ml, and most preferably no more than 2 ng / ml (e.g. Figure 1 shown).

[0282] In one embodiment, the present application provides an isolated murine TIGIT antibody that binds to CHOK1-TIGIT at a FACS binding EC50 of no more than 150 ng / ml, preferably no more than 100 ng / ml, more preferably no more than 90 ng / ml, more preferably no more than 80 ng / ml, more preferably no more than 60 ng / ml, further more preferably no more than 50 ng / ml, and most preferably no more than 30 ng / ml (e.g. Figure 2 shown).

[0283] In one embodiment, the present application provides an isolated murine TIGIT antibody that blocks TIGIT-mFc binding to CD155-hFC (e.g., TIGIT-mFc) with an ELISIA blocking IC50 of no more than 60 ng / ml, preferably no more than 50 ng / ml, more preferably no more than 48 ng / ml, more preferably no more than 45 ng / ml, more preferably no more than 40 ng / ml, further more preferably no more than 35 ng / ml, and most preferably no more than 30 ng / ml. Figure 3 shown).

[0284] In one embodiment, the present application provides an isolated murine TIGIT antibody that blocks CD155-hFc binding to CHOK1-TIGIT-52 (e.g., CD155-hFc binding to CHOK1-TIGIT-52) with a FACS blocking IC50 of no more than 200 ng / ml, preferably no more than 180 ng / ml, more preferably no more than 150 ng / ml, more preferably no more than 120 ng / ml, more preferably no more than 100 ng / ml, and even more preferably no more than 90 ng / ml. Figure 4 shown).

[0285] In one embodiment, the present application provides an isolated chimeric TIGIT antibody that binds to TIGIT-mFc (e.g., TIGIT-1 antibody) at an ELISIA binding EC50 of no more than 15 ng / ml, preferably no more than 12 ng / ml, more preferably no more than 1 ng / ml, more preferably no more than 10 ng / ml, more preferably no more than 8 ng / ml, further more preferably no more than 6 ng / ml, and most preferably no more than 5 ng / ml. Figure 5 shown).

[0286] In one embodiment, the present application provides an isolated chimeric TIGIT antibody that binds to CHOK1-TIGIT with a FACS binding EC50 of no more than 300 ng / ml, preferably no more than 258 ng / ml, more preferably no more than 220 ng / ml, more preferably no more than 180 ng / ml, further more preferably no more than 150 ng / ml, further more preferably no more than 100 ng / ml, further more preferably no more than 60 ng / ml (e.g. Figure 7 shown).

[0287] In one embodiment, the present application provides an isolated chimeric TIGIT antibody that blocks TIGIT binding to CD155 (e.g., CD155) at an ELISIA blocking IC50 of no more than 2080 ng / ml, preferably no more than 1500 ng / ml, more preferably no more than 1200 ng / ml, more preferably no more than 1000 ng / ml, further more preferably no more than 800 ng / ml, most preferably no more than 600 ng / ml, and most preferably no more than 500 ng / ml. Figure 6 shown).

[0288] In one embodiment, the present application provides an isolated chimeric TIGIT antibody that blocks the binding of CD155-hFc and CHOK1-TIGIT-52 with a FACS blocking activity of IC50 of no more than 200 ng / ml, preferably no more than 180 ng / ml, more preferably no more than 150 ng / ml, more preferably no more than 120 ng / ml, further more preferably no more than 100 ng / ml, and most preferably no more than 80 ng / ml (e.g. Figure 8 shown).

[0289] In one embodiment, in a Biacore assay, the chimeric TIGIT antibody of the present disclosure can specifically bind to a human TIGIT protein with a KD value of about no greater than 5E-09 M or less (e.g., the KD is no greater than about 9E-10 M, no greater than about 5E-10 M, no greater than about 4E-10 M, no greater than about 3E-10 M, or preferably no greater than about 4E-10 M). Fig. 9 , for example 25H8(464)-CHI.

[0290] In one embodiment, in a Biacore assay, the chimeric TIGIT antibody of the present disclosure can specifically bind to a human TIGIT protein with a KD value of about 1E-09 M or less (e.g., the KD is no more than about 9E-10 M, no more than about 8E-10 M, no more than about 5E-10 M, no more than about 6E-10 M, no more than about 5E-10 M, no more than about 3E-10, or no more than about 1E-10 M). Fig.10 As shown), for example BT-6A10-CHI.

[0291] In one embodiment, in a Biacore assay, the chimeric TIGIT antibody of the present disclosure can specifically bind to a human TIGIT protein with a KD value of about no greater than 1E-09 M or less (e.g., the KD is no greater than about 6E-10 M, no greater than about 5E-10 M, no greater than about 4E-10 M, no greater than about 3.5E-10 M, no greater than about 3.1E-10 M). Fig.11 As shown), for example BT-7F4-CHI.

[0292] In one embodiment, the present disclosure provides an isolated humanized anti-TIGIT antibody that binds to TIGIT (e.g., TIGIT 100) with an ELISA binding EC50 activity of no higher than 50 ng / ml, preferably no higher than 45 ng / ml, more preferably no higher than 40 ng / ml, more preferably no higher than 35 ng / ml, further preferably no higher than 30 g / ml, further more preferably no higher than 25 ng / ml, further more preferably no higher than 15 ng / ml, further more preferably no higher than 10 ng / ml, and most preferably no higher than 5 ng / ml. Fig.12 shown);.

[0293] In the present application, the isolated humanized anti-TIGIT humanized antibody blocks CD155 binding to CHOK1-TIGIT-52 protein (e.g., at a concentration of no more than 2500 ng / ml, or no more than 2000 ng / ml, or no more than 1800 ng / ml, or no more than 1500 ng / ml, or no more than 1300 ng / ml, or no more than 1200 ng / ml, or no more than 800 ng / ml and a FACS blocking IC50 of Fig.13 shown).

[0294] In the present application, the present disclosure provides an isolated humanized anti-TIGIT humanized antibody that binds to CHOK1-TIGIT-52 protein (e.g., TIGIT-52) with a FACS binding EC50 of no more than 300 ng / ml, or no more than 250 ng / ml, or no more than 200 ng / ml, or no more than 150 ng / ml, or no more than 100 ng / ml, or no more than 80 ng / ml, or no more than 50 ng / ml. Fig.14 shown).

[0295] In the present application, the isolated humanized anti-TIGIT humanized antibody blocks TIGIT binding to CD155 (e.g., CD155) with an ELISA blocking IC50 of no more than 300 ng / ml, or no more than 250 ng / ml, or no more than 200 ng / ml, or no more than 150 ng / ml, or no more than 100 ng / ml, or no more than 80 ng / ml, or no more than 50 ng / ml. Fig.15 (shown)

[0296] In one embodiment, in a Biacore assay, the protein can specifically bind to human TIGIT protein with a KD value of about 1E-09 M or less (e.g., the KD is no more than about 6E-10 M, no more than about 5E-10 M, no more than about 4E-10 M, no more than about 3.5E-10 M, no more than about 3.8E-10 M, such as Fig.16 , for example BT-6A10-31.

[0297] In one embodiment, in a Biacore assay, the protein can specifically bind to human TIGIT protein with a KD value of about no greater than 5E-09 M or less (e.g., the KD is no greater than about 9E-10 M, no greater than about 5E-10 M, no greater than about 4E-10 M, no greater than about 3E-10 M, or preferably no greater than E-10 M, such as Fig.17 , for example 25H8(464)-CHI.

[0298] In one embodiment, in a Biacore assay, the KD value can be no higher than about 5E-09M or less (for example, the KD is no higher than about 9E-10M, no higher than about 5E-10M, no higher than about 4E-10, no higher than about 3E-10M, or preferably no higher than E-10M) and specifically binds to human TIGIT protein. Fig.17 Medium, BT-25H8-41.

[0299] In one embodiment, in a Biacore assay, the protein can specifically bind to human TIGIT protein with a KD value of about no greater than 5E-09 M or less (e.g., the KD is no greater than about 9E-10 M, no greater than about 5E-10 M, no greater than about 4E-10 M, no greater than about 3E-10 M, or preferably no greater than E-10 M, such as Fig.18 , for example BT-6A10-33.

[0300] In one embodiment, in a Biacore assay, the protein can specifically bind to human TIGIT protein with a KD value of about no greater than 5E-09 M or less (e.g., the KD is no greater than about 9E-10 M, no greater than about 5E-10 M, no greater than about 4E-10 M, no greater than about 3E-10 M, or preferably no greater than E-10 M, such as Fig.19 , for example BT-6A10-32.

[0301] (II) Variable region

[0302] The VH and / or VL sequences (or CDR sequences) of other TIGIT antibodies that bind to human TIGIT can be "mixed and paired" with the VH and / or VL sequences (or CDR sequences) of the antibodies of the present application. Preferably, when VH and VL (or the CDRs therein) are mixed and paired, the VH sequence in a specific VH / VL pairing can be replaced by a structurally similar VH sequence. Similarly, it is preferred that the VL sequence in a specific VH / VL pairing is replaced by a structurally similar VL sequence.

[0303] On the one hand, the present application provides an isolated TIGIT antigen binding protein, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), which may comprise at least one CDR in the antibody heavy chain variable region VH.

[0304] In one embodiment, the present application provides an antibody or an antigen-binding portion thereof that specifically binds to TIGIT, comprising:

[0305] (a) comprises a heavy chain variable region selected from the VH amino acid sequences listed in Table 1, or a variant amino acid sequence having no less than 85% sequence identity with the VH amino acid sequences listed in Table 1; and

[0306] (b) comprises a light chain variable region selected from the VL amino acid sequences listed in Table 1, or a variant amino acid sequence having no less than 85% sequence identity with the VL amino acid sequences listed in Table 1.

[0307] Table 1 Heavy chain variable region and light chain variable region sequences of exemplary TIGIT antibodies of the present invention

[0308]

[0309]

[0310]

[0311] In some embodiments, the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 23, 25 or 27, or comprises a variant amino acid sequence with no less than 85% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 or 27. In the present application, the VHCDR of the isolated antigen-binding protein can be divided in any form, as long as VH is identical to the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 and 27, and the VHCDR obtained by dividing in any form can fall within the protection scope of the present application. In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 24, 26 or 28, or comprises a variant amino acid sequence with no less than 85% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 or 27. In the present application, the VHCDR of the isolated antigen-binding protein can be divided in any form, as long as VH is identical to the amino acid sequence shown in any one of SEQ ID NOs: 24, 26 or 28, and the VLCDR obtained by dividing in any form can fall within the protection scope of the present application. .

[0312] In one embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24. At least one exemplary embodiment is 25H8(464)-CHI.

[0313] In one embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO: 25, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26. At least one exemplary embodiment is 7F4(465)-CHI.

[0314] In one embodiment, the VH comprises the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28. At least one exemplary embodiment is 6A10(467)-CHI.

[0315] In some embodiments, the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 29-33, or comprises a variant amino acid sequence having no less than 85% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 29-33.

[0316] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 43-47, or comprises a variant amino acid sequence having no less than 85% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 43-47.

[0317] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 29, and the VL comprises the amino acid sequence shown in SEQ ID NO: 43. At least one exemplary embodiment is 25H8-1: H4A+L1A.

[0318] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 44. At least one exemplary embodiment is 25H8-2: H4B+L2A.

[0319] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 31, and the VL comprises the amino acid sequence shown in SEQ ID NO: 45. At least one exemplary embodiment is 25H8-3:H4C+L3A.

[0320] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises the amino acid sequence shown in SEQ ID NO: 46. At least one exemplary embodiment is 25H8-4: H4D+L6A.

[0321] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 47. At least one exemplary embodiment is 25H8-5: H2A+L1B.

[0322] In some embodiments, the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 34-37, or comprises a variant amino acid sequence having no less than 85% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 34-37.

[0323] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 48-51, or comprises a variant amino acid sequence having no less than 85% sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 48-51.

[0324] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO: 48. At least one exemplary embodiment is 7F4-1:H1A+L2A.

[0325] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO: 49. At least one exemplary embodiment is 7F4-2:H1A+L4A.

[0326] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 35, and the VL comprises the amino acid sequence shown in SEQ ID NO: 50. At least one exemplary embodiment is 7F4-3:H1B+L1A.

[0327] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51. At least one exemplary embodiment is 7F4-4:H7A+L2B.

[0328] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 37, and the VL comprises the amino acid sequence shown in SEQ ID NO: 48. At least one exemplary embodiment is 7F4-5:H3A+L2A.

[0329] On the one hand, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIGIT disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region (HCDRs) combination consisting of HCDR 1, HCDR 2, and HCDR3 and a light chain complementary determining region (LCDRs) combination consisting of LCDR 1, LCDR 2, and LCDR 3. In some embodiments, the VH comprises VHCDR1, VHCDR2, and VHCDR3 selected from those shown in Table 1, and the VL comprises VLCDR1, VLCDR2, and VLCDR3 selected from those shown in Table 1.

[0330] Table 1 Exemplary CDRs of anti-TIGIT antibodies of the present invention

[0331]

[0332] In one embodiment, the antibody or antigen binding portion thereof that specifically binds to TIGIT of the present application comprises VHCDR1, VHCDR2 and VHCDR3 and VLCDR1, VLCDR2 and VLCDR3:

[0333] (a) VHCDR1, VHCDR2 and VHCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, or SEQ ID NOs: 7, 8 and 9, or SEQ ID NOs: 13, 14 and 15, respectively, as listed in Table 2, or variant amino acids having no less than 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, or SEQ ID NOs: 7, 8 and 9, or SEQ ID NOs: 13, 14 and 15, as listed in Table 2; and

[0334] (b) VLCDR1, VLCDR2 and VLCDR3, respectively comprising the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, or SEQ ID NOs: 10, 11 and 12, or SEQ ID NOs: 16, 17 and 18 listed in Table 2, or variant amino acids having a sequence identity of not less than 85% with the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, or SEQ ID NOs: 10, 11 and 12, or SEQ ID NOs: 16, 17 and 18 listed in Table 2, or the CDR of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT.

[0335] In some embodiments, the heavy chain variable region CDR1-3 and the light chain variable region CDR4-6 of the anti-TIGIT antibody of the present invention comprise amino acid sequences having no less than 85% sequence homology, for example, 86%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100%, or any percentage sequence homology between any two adjacent values ​​of the above, with SEQ ID NOs: 1-6, respectively. In some embodiments, the heavy chain variable region CDR1-3 and the light chain variable region CDR4-6 of the anti-TIGIT antibody comprise amino acid sequences as shown in SEQ ID NOs: 1-6, respectively, and at least one exemplary embodiment is the murine antibody BT-25H8. At least one exemplary embodiment is the chimeric antibody BT-25H8 CHI. At least one exemplary embodiment is human antibodies 25H8-1: H4A+L1A, 25H8-2: H4B+L2A, 25H8-3: H4C+L3A, 25H8-4: H4D+L6A, and 25H8-5: H2A+L1B.

[0336] In some embodiments, the heavy chain variable region CDR1-3 and light chain variable region CDR1-3 of the anti-TIGIT antibody of the present invention comprise amino acid sequences having no less than 85% sequence homology with SEQ ID NO:7-12, respectively, for example, 86%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100%, or any percentage sequence homology between any two adjacent values ​​of the above.

[0337] In some embodiments, the heavy chain variable region CDR1-3 and the light chain variable region CDR4-6 of the anti-TIGIT antibody comprise SEQ ID NOs: 7-12, respectively. At least one exemplary embodiment is the murine antibody BT-7F4(465). At least one exemplary embodiment is the chimeric antibody BT-7F4(465)-CHI. At least one exemplary embodiment is the human antibodies 7F4-1:H1A+L2A, 7F4-2:H1A+L4A, 7F4-3:H1B+L1A, 7F4-4:H7A+L2B, and 7F4-5:H3A+L2A.

[0338] In some embodiments, the heavy chain variable region CDR1-3 and light chain variable region CDR1-3 of the anti-TIGIT antibody of the present invention comprise amino acid sequences having no less than 85% sequence homology with SEQ ID NOs: 13-18, for example, 86%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100%, or any percentage sequence homology between any two adjacent values ​​of the above.

[0339] In some embodiments, the heavy chain variable region CDR1-3 and the light chain variable region CDR4-6 of the anti-TIGIT antibody comprise SEQ ID NOs: 13-18, respectively. At least one exemplary embodiment is the murine antibody BT-6A10 (467). At least one exemplary embodiment is the chimeric BT-6A10 CHI. At least one exemplary embodiment is the human antibody 6A10-1: H3A+L1A, 6A10-2: H3B+L2A, 6A10-3: H3C+L4A, 6A10-4: H3D+L6A, 6A10-5: H3E+L3A.

[0340] Among them, the heavy chain variable region CDRs and the light chain variable region CDRs jointly determine the antibody's specific recognition and binding ability to the antigen. The mouse antibodies BT-7F4, BT-25H8 and BT-6A10 containing CDRs of SEQ ID NO: 1 to 6, or SEQ ID NO: 7 to 12 or SEQ ID NO: 13 to 18 have significant specific binding ability to TIGIT protein, and also have binding activity to TIGIT antigen expressed in cells. They can compete with CD155 for binding to TIGIT, and effectively block the binding of TIGIT and its ligand CD155 at the cellular level.

[0341] In the present invention, the complementarity determining region of the heavy chain is represented by HCDR or VHCDR, and the complementarity determining region of the light chain is represented by LCDR or VLCDR. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. In their compilation of "Sequences of Proteins of Immunological Interest", the amino acid sequences of light chain (λ, κ) variable regions and antibody heavy chains, as well as the variable regions of T cell receptors (α, β, γ, δ) are aligned and numbered. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention. In some embodiments, the mouse TIGIT antibodies disclosed in the present invention can be produced by hybridoma technology screening.

[0342] In another embodiment, the antibody or antigen-binding portion thereof of the present application includes the heavy chain variable region CDR2 of the TIGIT antibody and the CDRs of other antibodies that bind to human TIGIT, such as the heavy chain variable region CDR1, CDR2 and / or CDR3, and / or the light chain variable region CDR1, CDR2 and / or CDR3 of another TIGIT antibody.

[0343] In addition, it is well known in the art that the CDR3 domain, independent of CDR1 and / or CDR2, can determine the binding specificity of an antibody to the same antigen alone, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. See, for example, Klimka et al., British J. of Cancer 83 (2): 252-260 (2000); Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998), etc. These references are incorporated herein by reference in their entirety.

[0344] In some embodiments, the variable region modification is to mutate the amino acid residues in the VH and / or VL CDR1, CDR2 and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the target antibody. Point mutations or PCR-mediated mutations can be performed to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated in in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutation can be an amino acid replacement, addition or deletion, but preferably a replacement. In addition, no more than one, two, three, four or five residues in the CDR region are usually changed.

[0345] In addition, in another embodiment, the present application provides an isolated TIGIT monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, which comprises: (a) a VH CDR1 region, comprising a sequence of the present application, or an amino acid sequence obtained by substitution, deletion or addition of one, two, three, four or five amino acids thereof; (b) a VH CDR2 region, comprising a sequence of the present application, or an amino acid sequence obtained by substitution, deletion or addition of one, two, three, four or five amino acids thereof; (c) a VH CDR3 region, comprising a sequence of the present application, or an amino acid sequence obtained by substitution, deletion or addition of one, two, three, four or five amino acids thereof; (d) a VL CDR1 region, comprising a sequence of the present application, or an amino acid sequence obtained by substitution, deletion or addition of one, two, three, four or five amino acids thereof; (e) a VL CDR2 region, comprising a sequence of the present application, or an amino acid sequence obtained by substitution, deletion or addition of one, two, three, four or five amino acids thereof; and (f) a VL The CDR3 region comprises the sequence of the present application, or an amino acid sequence obtained by substitution, deletion or addition of one, two, three, four or five amino acids.

[0346] Genetically modified antibodies of the present application include those in which genetic modifications are made in the framework residues of VH and / or VL, for example, to change the antibody characteristics. Generally speaking, these framework modifications are used to reduce the immunogenicity of the antibody. For example, one method is to "revert" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that undergo somatic mutations may contain framework residues that are different from the germline sequence from which the antibody is obtained. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is obtained.

[0347] Another type of framework modification includes mutating one or more residues in the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the immunogenicity that may be caused by the antibody. This method is also called "deimmunization" and is described in more detail in U.S. Patent Publication 20030153043.

[0348] In addition, as another option outside of the modification in the framework or CDR region, the antibody of the present application can be genetically modified to include genetic modification in the Fc region, usually to change one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cellular toxicity. In addition, the antibody of the present application can be chemically modified (for example, one or more chemical functional groups can be added to the antibody), or modified to change its glycosylation to change one or more functional properties of the antibody.

[0349] In one embodiment, the hinge region of CH1 is modified to change, for example, increase or decrease the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Pat. No. 5,677,425. The cysteine ​​residues in the hinge region of CH1 are changed to, for example, promote the assembly of the heavy chain and light chain or increase / decrease the stability of the antibody.

[0350] In some non-limiting embodiments, the Fc hinge region of the TIGIT antibody of the present invention may be mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 connecting region of the Fc hinge fragment, so that the antibody has a weakened SpA binding relative to the native Fc-hinge domain SpA binding. This method is described in more detail in U.S. Patent 6,165,745.

[0351] In another embodiment, the antibody is modified by glycosylation. For example, a deglycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. For example, glycosylation modification can be achieved by, for example, replacing or modifying one or more amino acids to change one or more glycosylation sites in the antibody sequence to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that position to increase the affinity of the antibody for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0352] In another embodiment, the antibody of the present application comprises the CDR2 of the heavy chain variable region of the TIGIT antibody and at least the CDR3 of the heavy chain and / or light chain variable region of the TIGIT antibody, or the CDR3 of the heavy chain and / or light chain variable region of another TIGIT antibody, wherein the antibody is capable of specifically binding to human TIGIT. Preferably, these antibodies (a) compete for binding to TIGIT; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have a binding affinity similar to that of the TIGIT antibody of the present application.

[0353] In another embodiment, the antibody may further comprise the light chain variable region CDR2 of a TIGIT antibody, or the light chain variable region CDR2 of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT. In another embodiment, the antibody of the present application may comprise the heavy chain / light chain variable region CDR1 of a TIGIT antibody, or the heavy chain and / or light chain variable region CDR1 of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT.

[0354] In another embodiment, the antibody of the present application comprises a heavy chain and / or light chain variable region sequence or CDR1, CDR2 and CDR3 sequence that has one or more conservative modifications with the TIGIT antibody of the present application. It is known in the art that some conservative sequence modifications do not eliminate antigen binding. See, for example, Brummell et al., (1993) Biochem 32: 1180-8; deWildt et al., (1997) Prot. Eng. 10: 835-41; Adib-Conquy et al., (1998) Int. Immunol. 10: 341-6 and Beers et al., (2000) Clin. Can. Res. 6: 2835-43.

[0355] Therefore, in one embodiment, the TIGITD antibody of the present invention comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise HCDR1 HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, respectively, wherein:

[0356] (a) a HCDR1 of a heavy chain variable region comprising the HCDR1 sequence listed in Table 2, and / or conservative modifications thereof; and / or

[0357] (b) a HCDR2 of a heavy chain variable region comprising the HCDR2 sequence listed in Table 2, and / or conservative modifications thereof; and / or

[0358] (c) a heavy chain variable region HCDR3 comprising the HCDR3 sequence listed in Table 2, and / or conservative modifications thereof; and / or

[0359] (d) a light chain variable region LCDR1, and / or LCDR2, and / or LCDR3 comprising the LCDR1, LCDR2 and LCDR3 sequences listed in Table 2, and / or conservative modifications thereof; and

[0360] (e) The antibody specifically binds to human TIGIT.

[0361] The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid replacement, addition and deletion. Modifications can be introduced into the present application antibody by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacement is the replacement of amino acid residues with amino acid residues having similar side chains. Amino acid residue groups with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of the antibody of the present application can be replaced with other amino acid residues in the same side chain group, and the resulting antibody can be tested for retention of function (ie, the function described above) using the functional assays described herein.

[0362] Genetically modified antibodies

[0363] The antibody of the present application can be prepared by having one or more V H / V L The antibody sequence is used as the starting material to prepare the genetically modified antibody. The antibody can be prepared by modifying one or two variable regions (i.e., V H and / or V L ) (e.g., in one or more CDR regions and / or one or more framework regions) to improve binding affinity and / or increase similarity to antibodies naturally produced by certain species. For example, the framework regions are modified to provide humanized antibodies. In addition, or antibodies can be genetically modified by modifying residues in the constant regions, for example, to alter the effector function of the antibody.

[0364] In some embodiments, CDR regions are implanted into variable regions that can be used to genetically modify antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementary determining regions (CDRs). For this reason, the amino acid residues within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because the CDR sequence is responsible for the main antibody-antigen interaction, recombinant antibodies that mimic the properties of specific natural antibodies can be expressed by constructing an expression vector containing the CDR sequence of a specific natural antibody implanted into the framework sequence of a different antibody with different properties (Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al.. (1989) Proc. Natl. Acad; USA 86:10029-10033; US Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0365] Therefore, another embodiment of the present application relates to an isolated monoclonal antibody or an antigen-binding portion thereof, which comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 having the above-mentioned sequence of the present application, and the light chain variable region comprises CDR1, CDR2 and CDR3 having the above-mentioned sequence of the present application. Although these antibodies comprise the VHCDR and VLCDR sequences of the monoclonal antibodies of the present application, they may contain different framework sequences.

[0366] Such framework sequences can be obtained from public DNA databases or public references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk / vbase) and Kabat et al., (1991), supra; Tomlinson et al., (1992) J. Mol. Biol. 227: 776-798; and Cox et al., (1994) Eur. J. Immunol. 24: 827-836. As another embodiment, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database.

[0367] The antibody protein sequence can be compared with a protein sequence database by using one of the sequence similarity search methods known in the art, called gap BLAST (Altschul et al., (1997)). Preferred framework sequences for the antibodies of the present application are those structurally similar to the framework sequences used for the antibodies of the present application. The VHCDR1, VHCDR2, and VHCDR3 sequences can be implanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence was obtained, or the CDR sequence can be implanted into a framework region containing one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate the residues in the framework region to maintain or enhance the antigen binding of the antibody (see, e.g., US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0368] Through the aforementioned gene transformation and modification, exemplary heavy chain framework regions and light chain framework regions of the TIGIT monoclonal antibodies obtained in the present invention are shown in Tables 3 and 4, respectively. The heavy chain framework region comprises VH-FR1, VH-FR2, VH-FR3 and VH-FR4, and the light chain framework region comprises VL-FR1, VL-FR2, VL-FR3 and VL-FR4.

[0369] Table 3 Exemplary framework regions of the TIGIT monoclonal antibodies of the present invention Heavy chain framework regions

[0370]

[0371]

[0372]

[0373] Table 4 Exemplary framework regions of TIGIT monoclonal antibodies of the present invention Light chain framework regions.

[0374]

[0375]

[0376] In the present application, the TIGIT antigen binding protein may include a light chain variable region VH, and the VH may include at least one, at least two or at least three of HCDR1, HCDR2 and HCDR3. At least one CDR in the light chain variable region VH of the anti-TIGIT antibody, for example, the VH may include an amino acid sequence shown in any one of SEQ ID NOs: 23, 25 or 27; for example, the VH of the antigen binding protein may include an amino acid sequence shown in any one of SEQ ID NOs: 29-42. In the present application, the LCDR of the isolated antigen binding protein may be divided in any form, as long as the VH is the same as the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 or 27 or SEQ ID NOs: 29-42, and the LCDR obtained by dividing in any form may fall within the protection scope of the present application.

[0377] In the present application, the HCDR1 of the TIGIT antigen binding protein may comprise the amino acid sequences shown in SEQ ID NOs: 1, 7 and 13. For example, the LCDR1 of the antigen binding protein may be defined according to the Kabat numbering system.

[0378] In the present application, the HCDR2 of the TIGIT antigen binding protein may comprise the amino acid sequences shown in SEQ ID NOs: 2, 8 and 14. For example, the HCDR2 of the antigen binding protein may be defined according to the Kabat numbering system.

[0379] In the present application, the LCDR3 of the TIGIT antigen binding protein may comprise the amino acid sequence shown in SEQ ID NOs: 3, 9 and 15. For example, the HCDR3 of the antigen binding protein may be defined according to the Kabat numbering system. In one embodiment of the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 91-94. In one embodiment of the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 95-98. In one embodiment of the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 99-104. For example, the H-FR1 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0380] In one embodiment of the present application, the H-FR2 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 105-108. In one embodiment of the present application, the H-FR2 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 109-111. In one embodiment of the present application, the H-FR2 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 112-113. For example, the H-FR2 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0381] In one embodiment of the present application, the H-FR3 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 114-117. In one embodiment of the present application, the H-FR3 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 118-121. In one embodiment of the present application, the H-FR3 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 122-124. For example, the H-FR3 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0382] In one embodiment of the present application, the H-FR4 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 125-128. In one embodiment of the present application, the H-FR4 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 129-131. In one embodiment of the present application, the H-FR4 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 132-135. For example, the H-FR4 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0383] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 91-94; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 105-108; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 114-117; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 125-128. For example, the antigen binding protein may include the antibody BT-25H8-CHII or an antibody having the same H-FR1-4.

[0384] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 91; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 105; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 145; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 125. For example, the antigen binding protein may include the antibody BT-25H8-CHI or an antibody having the same H-FR1-4.

[0385] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 92; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 106; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 115; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 126. For example, the antigen binding protein may include antibody 25H8-1: H4A+L1A or an antibody having the same H-FR1-4.

[0386] In one embodiment of the present application, the H-FR1 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 93; the H-FR2 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 107; the H-FR3 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 116; and the H-FR4 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 127. For example, the antigen-binding protein may include antibody 25H8-2: H4B+L2A or an antibody having the same H-FR1-4.

[0387] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 93; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 107; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 116; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 126. For example, the antigen binding protein may include antibody 25H8-3:H4C+L3A or an antibody having the same H-FR1-4.

[0388] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 93; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 107; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 116; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 128. For example, the antigen binding protein may include antibody 25H8-4: H4D+L6A or an antibody having the same H-FR1-4.

[0389] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 94; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 108; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 117; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 128. For example, the antigen binding protein may include antibody 25H8-5: H2A+L1B or an antibody having the same H-FR1-4.

[0390] In one embodiment of the present application, the H-FR1 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 95-98; the H-FR2 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 109-111; the H-FR3 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 118-121; and the H-FR4 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 129-130. For example, the antigen-binding protein may include the antibody BT-7F4-CHI or an antibody having the same H-FR1-4.

[0391] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 95; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 109; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 118; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 129. For example, the antigen binding protein may include the antibody BT-7F4-CHI or an antibody having the same H-FR1-H-FR4.

[0392] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 96; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 110; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 119; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 130. For example, the antigen binding protein may include antibodies 7F4-1: H1A + L2A, 7F4-2: H1A + L4A, or antibodies having the same H-FR1-H-FR4.

[0393] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 96; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 110; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 119; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 131. For example, the antigen binding protein may include antibody 7F4-3: H1B+L1A or an antibody having the same H-FR1-H-FR4.

[0394] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 97; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 110; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 120; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 131. For example, the antigen binding protein may include antibody 7F4-4: H7A+L2B or an antibody having the same H-FR1-H-FR4.

[0395] In one embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 98; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 111; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 121; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 130. For example, the antigen binding protein may include antibody 7F4-5: H3A+L2A or an antibody having the same H-FR1-H-FR4. In some embodiments of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NOs: 99-104; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NOs: 112-113; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NOs: 122-124; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NOs: 132-135. For example, the antigen binding protein may include antibodies 6A10(467)-CHI, BT-6A10-31, BT-6A10-34, BT-6A10-36 and BT-6A10-33 or antibodies having the same H-FR1-4 thereof. In an exemplary embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 99; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 112; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 122; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 132. For example, the antigen binding protein may include antibody BT-6A10(467)-CHI or an antibody having the same H-FR1-4 thereof.

[0396] In an exemplary embodiment of the present application, the H-FR1 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 100; the H-FR2 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 113; the H-FR3 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 123; and the H-FR4 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NO: 133. For example, the antigen-binding protein may include the antibody BT-6A10-1: H3A+L1A or an antibody having the same H-FR1-4.

[0397] In an exemplary embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 101; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 123; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 133. For example, the antigen binding protein may include antibody 6A10-2: H3B+L2A or an antibody having the same H-FR1-4.

[0398] In an exemplary embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 102; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 123; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 134. For example, the antigen binding protein may include antibody 6A10-3: H3C+L4A or an antibody having the same H-FR1-4.

[0399] In an exemplary embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 103; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 123; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 135. For example, the antigen binding protein may include antibody 6A10-4: H3D+L6A or an antibody having the same H-FR1-4.

[0400] In an exemplary embodiment of the present application, the H-FR1 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 104; the H-FR2 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 124; and the H-FR4 of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 133. For example, the antigen binding protein may include antibody 6A10-5: H3E+L3A or an antibody having the same H-FR1-4.

[0401] In the present application, the TIGIT antigen binding protein may include a light chain variable region VL, and the VL may include at least one, at least two or at least three of LCDR1, LCDR2 and LCDR3. At least one CDR in the light chain variable region VL of the anti-TIGIT antibody, for example, the VL may include an amino acid sequence shown in any one of SEQ ID NOs: 24, 26 or 28; for example, the VL of the antigen binding protein may include an amino acid sequence shown in any one of SEQ ID NOs: 43-56. In the present application, the LCDR of the isolated antigen binding protein may be divided in any form, as long as the VL is the same as the amino acid sequence shown in SEQ ID NOs: 24, 26 or 28 or any one of SEQ ID NOs: 43-56, and the LCDR obtained by dividing in any form may fall within the protection scope of the present application.

[0402] In the present application, the LCDR1 of the TIGIT antigen binding protein may comprise the amino acid sequences shown in SEQ ID NOs: 4, 10 and 16. For example, the LCDR1 of the antigen binding protein may be defined according to the Kabat numbering system.

[0403] In the present application, the LCDR2 of the TIGIT antigen binding protein may comprise the amino acid sequences shown in SEQ ID NOs: 5, 11 and 17. For example, the LCDR2 of the antigen binding protein may be defined according to the Kabat numbering system.

[0404] In the present application, the LCDR3 of the TIGIT antigen binding protein may comprise the amino acid sequences shown in SEQ ID NOs: 6, 12 and 18. For example, the LCDR3 of the antigen binding protein may be defined according to the Kabat numbering system.

[0405] For example, the VL of the TIGIT antigen binding protein may comprise framework regions L-FR1, L-FR2, L-FR3 and L-FR4.

[0406] In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 136-150.

[0407] In the present application, the L-FR1 of the antigen binding protein may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 136-140, 141-144, or 145-150. For example, the L-FR1 of the antigen binding protein may be defined according to the Kabat numbering system.

[0408] In the present application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NOs: 151-155. In the present application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 156-159. In the present application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 160-165. For example, the L-FR2 of the antigen binding protein may be defined according to the Kabat numbering system.

[0409] In the present application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 166-180. In the present application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 166-170 or SEQ ID NOs: 171-174. In the present application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 175-180. For example, the L-FR3 of the antigen binding protein may be defined according to the Kabat numbering system.

[0410] In the present application, the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 181 to 185. For example, the L-FR4 of the antigen binding protein may be defined according to the Kabat numbering system.

[0411] In the present application, the L-FR1 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 136-140; the L-FR2 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 151-154; the L-FR3 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 166-170; the L-FR4 of the antigen-binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 181-185. For example, the antigen-binding protein may include antibodies BT-25H8-CHI, BT-25H8-41, 25H8-2: H4B+L2A, 25H8-3: H4C+L3A, 25H8-4: H4D+L6A, 25H8-5: H2A+L1B, or antibodies having the same LCDR3 (e.g., having the same LCDR1-3) therewith.

[0412] In the present application, the L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 141-144; the L-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 156-159; the L-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 171-174; and the L-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 181-185. For example, the antigen-binding protein may include antibodies 7F4(465)-CHI, 7F4-1: H1A+L2A, 7F4-2: H1A+L4A, 7F4-3: H1B+L1A, 7F4-4: H7A+L2B, 7F4-5: H3A+L2A, or antibodies having the same LCDR3 (e.g., having the same LCDR1-3) therewith.

[0413] In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 145-150; the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 160-165; the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 175-180; and the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 181-184. For example, the antigen binding protein may include antibodies 6A10(467)-CHI, 6A10-1: H3A+L1A, 6A10-2: H3B+L2A, 6A10-3: H3C+L4A, or 6A10-4: H3D+L6A, 6A10-5: H3E+L3A and antibodies having the same LCDR3 (e.g., having the same LCDR1-3) as the antibodies.

[0414] In one embodiment, the VH-FR1 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 91-104, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 91-104 having no less than 85% sequence identity. In one embodiment, the VH-FR2 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 105-113, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 105-113 having no less than 85% sequence identity. In one embodiment, the VH-FR3 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 114-124, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 114-124 having no less than 85% sequence identity. In one embodiment, the VH-FR4 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 125-135, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 125-135 having no less than 85% sequence identity. In one embodiment, the VL-FR1 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 136-150, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 136-150 having no less than 85% sequence identity. In one embodiment, the VL-FR2 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 151-165, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 151-165 having no less than 85% sequence identity. In one embodiment, the VL-FR3 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 166-180, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 166-180 having no less than 85% sequence identity. In one embodiment, the VL-FR4 of the present invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 181-185, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 181-185 having no less than 85% sequence identity.

[0415] (ii) Constant region

[0416] In the present application, the TIGIT antibody or antigen-binding protein further comprises a heavy chain constant region.

[0417] For example, the antigen binding protein may include a constant region derived from IgG. For example, the antigen binding protein may include a heavy chain. Wherein the heavy chain may include a heavy chain variable region and a heavy chain constant region. The heavy chain variable region and the heavy chain constant region may be directly or indirectly connected. For example, the heavy chain variable region may be directly connected to the heavy chain constant region. For example, the heavy chain constant region may include a constant region derived from a protein selected from the group consisting of: IgG1, IgG2, IgG3 and IgG4. In some embodiments, the species of the heavy chain constant region and the light chain constant region are human IgG1 or human IgG4. In some embodiments, the species of the heavy chain constant region is human IgG4. For example, the heavy chain constant region of the binding protein of the antibody that specifically binds to TIGIT may include the amino acid sequence shown in SEQ ID NO: 222.

[0418] In the present application, the anti-TIGIT antibody or antigen binding protein may comprise a light chain variable region and a light chain constant region. Wherein, the light chain variable region and the light chain constant region may be directly or indirectly connected. For example, the light chain constant region may be a κ chain or a λ chain. For example, the light chain constant region of the binding protein of the antibody that specifically binds to the TIGIT antigen may comprise an amino acid sequence as shown in SEQ ID NO: 223.

[0419] (III) Full-length heavy and light chains

[0420] On the one hand, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to TIGIT, which is a full-length antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC and LC comprise the amino acid sequences of HCH and LC as shown in Table 5.

[0421] In one embodiment, the HC comprises the amino acid sequence shown in any one of SEQ ID NOs: 57-73, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 57-73; or in one embodiment, the LC comprises the amino acid sequence shown in any one of SEQ ID NOs: 74-90, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 74-90.

[0422] Exemplary heavy chains and light chains of the TIGIT monoclonal antibodies of the present invention are shown in Table 5, respectively.

[0423]

[0424]

[0425]

[0426]

[0427]

[0428] In one embodiment of the TIGIT antibody of the present invention, the HC comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-73, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-73 that has complementary 85% sequence identity with the amino acid sequence.

[0429] In one embodiment of the TIGIT antibody of the present invention, the LC comprises an amino acid sequence selected from any one of SEQ ID NOs: 74-90, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 74-90 that has complementary 85% sequence identity with the amino acid sequence.

[0430] In one embodiment of the TIGIT antibody of the present invention, the HC comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-62, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 57-62 having a complementary region of 85% sequence identity. In one embodiment of the TIGIT antibody of the present invention, the LHC comprises an amino acid sequence selected from any one of SEQ ID NOs: 74-79, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 74-79 having a complementary region of 85% sequence identity. In some exemplary embodiments, the TIGIT antibody is a chimeric antibody 25H8(464)-CHI. In some exemplary embodiments, the TIGIT antibody is a humanized antibody 25H8-1:H4A+L1A(25H8-41). In some exemplary embodiments, the TIGIT antibody is a humanized antibody 25H8-2:H4B+L2A(25H8-42).

[0431] In one embodiment of the TIGIT antibody of the present invention, the HC comprises an amino acid sequence selected from any one of SEQ ID NOs: 63-67, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 63-67 having a complementary region of 85% sequence identity. In one embodiment of the TIGIT antibody of the present invention, the LHC comprises an amino acid sequence selected from any one of SEQ ID NOs: 80-81, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 80-81 having a complementary region of 85% sequence identity. In some exemplary embodiments, the TIGIT antibody is a chimeric antibody 7F4(465)-CHI. In some exemplary embodiments, the TIGIT antibody is a humanized antibody 7F4-1:H1A+L2A(7F4-12)). In some exemplary embodiments, the TIGIT antibody is a humanized antibody 7F4-2:H1A+L4A(7F4-14). In some exemplary embodiments, the TIGIT antibody is the humanized antibody 7F4-3:H1B+L1A (7F4-31).

[0432] In one embodiment of the TIGIT antibody of the present invention, the HC comprises an amino acid sequence selected from any one of SEQ ID NOs: 68-73, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 63-67 having a complementary region with 85% sequence identity. In one embodiment of the TIGIT antibody of the present invention, the LHC comprises an amino acid sequence selected from any one of SEQ ID NOs: 85-90, or comprises an amino acid sequence selected from any one of SEQ ID NOs: 85-90 having a complementary region with 85% sequence identity. In some exemplary embodiments, the TIGIT antibody is a chimeric antibody 6A10(467)-CHI. In some exemplary embodiments, the TIGIT antibody is a humanized antibody 6A10-1: H3A+L1A (6A104-31). In some exemplary embodiments, the TIGIT antibody is a humanized antibody 6A10-2: H3B+L2A (6A104-32). In some exemplary embodiments, the TIGIT antibody is a humanized antibody 6A10-3: H3C + L4A (6A104-34). In some exemplary embodiments, the TIGIT antibody is a humanized antibody 6A10-5: H3E + L3A (6A104-33).

[0433] (IV) Exemplary Antibodies

[0434] In one embodiment of the TIGIT antibody of the present invention, the heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO: 1, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 3 in Table 2; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 4, the light chain CDR2 shown in SEQ ID NO: 5, and the light chain CDR3 shown in SEQ ID NO: 6;

[0435] Wherein, in at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 23 in Table 1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24 in Table 1;

[0436] Wherein, in at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 91, 105, 114 and 125 in Table 3, respectively, and the light chain variable region of the anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 136, 151, 166 and 181 in Table 4, respectively;

[0437] The heavy chain variable region of the anti-TIGIT monoclonal antibody and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain.

[0438] In at least one specific embodiment, preferably, the heavy chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 57 in Table 5; the light chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 74 in Table 5.

[0439] In at least one embodiment, the anti-TIGIT antibody is a chimeric antibody 25H8(464)-CHI.

[0440] In one embodiment, the anti-TIGIT antibody heavy chain variable region includes a heavy chain CDR1 as shown in SEQ ID NO:7 in Table 2, a heavy chain CDR2 as shown in SEQ ID NO:8, and a heavy chain CDR3 as shown in SEQ ID NO:9; the anti-TIGIT antibody light chain variable region includes a light chain CDR1 as shown in SEQ ID NO:10 in Table 2, a light chain CDR2 as shown in SEQ ID NO:11, and a light chain CDR3 as shown in SEQ ID NO:12.

[0441] In at least one specific embodiment, the heavy chain variable region of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO: 25 in Table 1; the light chain variable region of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO: 26 in Table 1.

[0442] In at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 95, 109, 118 and 129 in Table 3, respectively, and the light chain variable region of the anti-TIGIT anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 141, 156, 171 and 181 in Table 4, respectively.

[0443] Among them, in at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT monoclonal antibody and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQID NO: 223) are connected to form a light chain.

[0444] In at least one specific embodiment, preferably, the heavy chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:63 in Table 5; the light chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:80 in Table 5.

[0445] Among them, in at least one preferred embodiment, the anti-TIGIT antibody is a chimeric antibody 7F4(465)-CHI.

[0446] In one embodiment, the heavy chain variable region of the anti-TIGIT antibody includes a heavy chain CDR1 as shown in SEQ ID NO:13 in Table 2, a heavy chain CDR2 as shown in SEQ ID NO:14, and a heavy chain CDR3 as shown in SEQ ID NO:15; the light chain variable region of the anti-TIGIT antibody includes a light chain CDR1 as shown in SEQ ID NO:16 in Table 2, a light chain CDR2 as shown in SEQ ID NO:17, and a light chain CDR3 as shown in SEQ ID NO:18.

[0447] Among them, in at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT antibody includes the amino acid sequence shown in SEQ ID NO: 27 in Table 1; the light chain variable region of the anti-TIGIT antibody includes the amino acid sequence shown in SEQ ID NO: 28 in Table 1.

[0448] Among them, in at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 99, 112, 122 and 132 in Table 3, respectively, and the light chain variable region of the anti-TIGIT antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 145, 160, 175 and 181 in Table 3, respectively.

[0449] Among them, in at least one specific embodiment, preferably, the heavy chain variable region of the anti-TIGIT antibody and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain.

[0450] In at least one specific embodiment, preferably, the heavy chain of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO:68 in Table 5; the light chain of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO:85 in Table 5.

[0451] In at least one embodiment, the anti-TIGIT chimeric antibody is 6A10(467)4-CHI.

[0452] In some embodiments, the anti-TIGIT monoclonal antibody is a humanized monoclonal antibody.

[0453] In some embodiments, the humanized monoclonal antibody heavy chain variable region VHCDR1-3 and light chain variable region VLCDR1-3 jointly determine the antibody's specific recognition and binding ability to the antigen. The mouse antibodies BT-7F4, BT-25H8 and BT-6A10 containing CDRs of SEQ ID NOs: 1-6, or SEQ ID NOs: 7-12 or SEQ ID NOs: 13-18 have significant specific binding ability to TIGIT protein, and also have binding activity to TIGIT antigen expressed by cells, can compete with CD155 for binding to TIGIT, and effectively block the binding of TIGIT and its ligand CD155 at the cellular level.

[0454] In some embodiments, in the humanized anti-TIGIT monoclonal antibody of the present invention, the VH comprises HC-CDR1 shown in SEQ ID NO: 1, 7 or 13, HC-CDR2 shown in SEQ ID NO: 2, 8 or 14, and HC-CDR3 shown in SEQ ID NO: 3, 9 or 15;

[0455] In some embodiments, the humanized anti-TIGIT monoclonal antibody of the present invention, the VL includes LC-CDR1 shown in SEQ ID NO:4, 10 or 16, LC-CDR2 shown in SEQ ID NO:5, 11 or 17, and LC-CDR3 shown in SEQ ID NO:6, 12 or 18.

[0456] In some embodiments, in the humanized anti-TIGIT monoclonal antibody of the present invention, the VH comprises HC-CDR1 shown in SEQ ID NO: 1 in Table 2, HC-CDR2 shown in SEQ ID NO: 2, and HC-CDR3 shown in SEQ ID NO: 3; the VL comprises LC-CDR1 shown in SEQ ID NO: 4 in Table 2, LC-CDR2 shown in SEQ ID NO: 5, and LC-CDR3 shown in SEQ ID NO: 6;

[0457] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 29 in Table 1; the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 43 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NO: 92, 106, 115 and 126 in Table 3, respectively, and the light chain variable region of the anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NO: 137, 152, 167 and 182 in Table 4, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is connected to the human IgG4 heavy chain constant region (SEQ ID NO: 222) to form a heavy chain, and the light chain variable region is connected to the human κ light chain constant region (SEQ ID NO: 223) to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 58 in Table 5, and the light chain includes the amino acid sequence shown in SEQ ID NO: 75. In at least one embodiment, the humanized anti-TIGIT monoclonal antibody is 25H8-1: H4A + L1A.

[0458] In some embodiments, the heavy chain variable region of the humanized anti-TIGIT antibody includes the heavy chain CDR1 shown in SEQ ID NO: 1 in Table 2, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 3; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 4 in Table 2, the light chain CDR2 shown in SEQ ID NO: 5, and the light chain CDR3 shown in SEQ ID NO: 6; wherein in at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 30 in Table 1; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 44 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 93, 107, 116 and 127 in Table 3, respectively, and the light chain variable region of the anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 138, 153, 168 and 183 in Table 1, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 59 in Table 5; the light chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 76 in Table 5. In at least one embodiment, the humanized anti-TIGIT antibody is 25H8-2:H4B+L2A (25H8-42).

[0459] In one embodiment, the heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO: 1 in Table 2, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 3; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 4 in Table 2, the light chain CDR2 shown in SEQ ID NO: 5, and the light chain CDR3 shown in SEQ ID NO: 6.

[0460] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:31 in Table 1; the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:45,

[0461] In at least one embodiment, preferably, the heavy chain variable region of the anti-humanized TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 with amino acid sequences shown as SEQ ID NO:93, 107, 116 and 126 in Table 3, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 with amino acid sequences shown as SEQ ID NO:139, 154, 169 and 183 in Table 4, respectively.

[0462] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 60 in Table 5; the light chain of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 77 in Table 5. In at least one embodiment, the humanized anti-TIGIT antibody is 25H8-3: H4C + L3A.

[0463] In one embodiment of the humanized anti-TIGIT monoclonal antibody of the present invention, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody includes the heavy chain CDR1 shown in SEQ ID NO: 1, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 3 in Table 2; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 4, the light chain CDR2 shown in SEQ ID NO: 5, and the light chain CDR3 shown in SEQ ID NO: 6 in Table 2. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 32 in Table 1; the light chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 46 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 93, 107, 116 and 128, respectively, in Table 3, and the light chain variable region of the anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 140, 155, 170 and 183, respectively, in Table 4. In at least one embodiment, preferably, the heavy chain variable region of the anti-TIGIT monoclonal antibody is connected to anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 219) to form a heavy chain, and the light chain variable region is connected to the human κ light chain constant region (SEQ ID NO: 220) to form a light chain. In at least one embodiment, preferably, the heavy chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 61 in Table 5; the light chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 78 in Table 5. In at least one embodiment, the humanized anti-TIGIT antibody is 25H8-4: H4D+L6A.

[0464] In one embodiment of the humanized anti-TIGIT monoclonal antibody of the present invention, the heavy chain variable region of the humanized anti-TIGIT antibody includes the heavy chain CDR1 shown in SEQ ID NO: 1, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 3 in Table 2; the light chain variable region of the humanized anti-TIGIT antibody includes the light chain CDR1 shown in SEQ ID NO: 4, the light chain CDR2 shown in SEQ ID NO: 5, and the light chain CDR3 shown in SEQ ID NO: 6 in Table 2. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 33 in Table 1; the light chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 47 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 94, 108, 117 and 28 in Table 3, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 137, 152, 167 and 184 in Table 4, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is connected to the human IgG4 heavy chain constant region (SEQ ID NO: 222) to form a heavy chain, and the light chain variable region is connected to the human κ light chain constant region (SEQ ID NO: 223) to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 62; and the light chain of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 79. In at least one embodiment, the humanized anti-TIGIT antibody is 25H8-5:H2A+L1B.

[0465] In one embodiment of the humanized anti-TIGIT monoclonal antibody of the present invention, the heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO: 7, the heavy chain CDR2 shown in SEQ ID NO: 8, and the heavy chain CDR3 shown in SEQ ID NO: 9; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO: 10, the light chain CDR2 shown in SEQ ID NO: 11, and the light chain CDR3 shown in SEQ ID NO: 12. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 34 in Table 1; the light chain variable region of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 48 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 with amino acid sequences shown as SEQ ID NOs: 96, 110, 119 and 130 in Table 5, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 with amino acid sequences shown as SEQ ID NOs: 142, 157, 171 and 184, respectively.

[0466] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 64; the light chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 81. In at least one embodiment, the humanized anti-TIGIT antibody is 7F4-1: H1A + L2A.

[0467] In one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 34 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 49 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs: 96, 110, 119 and 130, respectively, in Table 5, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs: 143, 158, 173 and 171, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 219) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 220) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 64; the light chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 82. In at least one embodiment, the humanized anti-TIGIT antibody is 7F4-2: H1A + L4A.

[0468] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 35 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 50 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs: 96, 110, 119 and 131 in Table 5, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs: 144, 159, 179 and 185, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 219) are connected to form a heavy chain, and the humanized light chain variable region and the human κ light chain constant region (SEQ ID NO: 220) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 65; the light chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 83. In at least one embodiment, the humanized anti-TIGIT antibody is 7F4-3: H1B + L1A.

[0469] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:36 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:51 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs:97, 110, 120 and 131 in Table 5, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs:142, 157, 172 and 182, respectively. In at least one embodiment, preferably, the heavy chain variable region of the anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 66; the light chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 84. In at least one embodiment, the humanized anti-TIGIT antibody is 7F4-4: H7A + L2B.

[0470] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 37 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 48 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs: 98, 111, 121 and 130, respectively, in Table 5, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs: 142, 157, 172 and 182, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 67; the light chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 81. In at least one embodiment, the humanized anti-TIGIT antibody is 7F4-5: H3A + L2A.

[0471] In one embodiment of the humanized anti-TIGIT monoclonal antibody of the present invention, the heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO:13, the heavy chain CDR2 shown in SEQ ID NO:14, and the heavy chain CDR3 shown in SEQ ID NO:15; the light chain variable region includes the light chain CDR1 shown in SEQ ID NO:16, the light chain CDR2 shown in SEQ ID NO:17, and the light chain CDR3 shown in SEQ ID NO:18.

[0472] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 38; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 52. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 with amino acid sequences shown in SEQ ID NOs: 100, 113, 123 and 133, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 with amino acid sequences shown in SEQ ID NOs: 146, 161, 176 and 182, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is connected to the human IgG4 heavy chain constant region (SEQ ID NO: 222) to form a heavy chain, and the light chain variable region is connected to the human κ light chain constant region (SEQ ID NO: 223) to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 69; the light chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 86. In at least one embodiment, the humanized anti-TIGIT antibody is 6A10-1: H3A + L1A.

[0473] In one embodiment of the humanized anti-TIGIT monoclonal antibody, the heavy chain variable region includes a heavy chain CDR1 as shown in SEQ ID NO: 13 in Table 2, a heavy chain CDR2 as shown in SEQ ID NO: 14, and a heavy chain CDR3 as shown in SEQ ID NO: 15; the light chain variable region includes a light chain CDR1 as shown in SEQ ID NO: 16 in Table 2, a light chain CDR2 as shown in SEQ ID NO: 17, and a light chain CDR3 as shown in SEQ ID NO: 18. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody includes an amino acid sequence as shown in SEQ ID NO: 39 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody includes an amino acid sequence as shown in SEQ ID NO: 53 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 of the amino acid sequences shown in SEQ ID NOs: 101, 113, 123 and 133, respectively, in Table 5, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 of the amino acid sequences shown in SEQ ID NOs: 147, 162, 177 and 182, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 222) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 223) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 70; and the light chain of the anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 87. In at least one embodiment, the humanized anti-TIGIT antibody is 6A10-2:H3B+L2A.

[0474] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 40 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 54 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs: 102, 113, 123 and 134, respectively, in Table 5, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs: 148, 163, 178 and 182, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 219) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 220) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 71; the light chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 88. In at least one embodiment, the humanized anti-TIGIT antibody is 6A10-3: H3C + L4A.

[0475] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:41 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:55 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs:103, 113, 123 and 135, respectively, in Table 5, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs:149, 164, 179 and 182, respectively. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 219) are connected to form a heavy chain, and the humanized light chain variable region and the human κ light chain constant region (SEQ ID NO: 222) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 72; the light chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 89. In at least one specific embodiment, the humanized anti-TIGIT antibody is 6A10-4: H3D + L6A.

[0476] In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 42 in Table 1; and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 56 in Table 1. In at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody comprises HC-FR1, HC-FR2, HC-FR3 and HC-FR4 having amino acid sequences shown in SEQ ID NOs: 104, 113, 124 and 133 in Table 5, respectively, and the light chain variable region of the humanized anti-TIGIT monoclonal antibody comprises LC-FR1, LC-FR2, LC-FR3 and LC-FR4 having amino acid sequences shown in SEQ ID NOs: 150, 165, 180 and 184, respectively. Among them, in at least one embodiment, preferably, the heavy chain variable region of the humanized anti-TIGIT monoclonal antibody is anti-TIGIT and the human IgG4 heavy chain constant region (SEQ ID NO: 219) are connected to form a heavy chain, and the light chain variable region and the human κ light chain constant region (SEQ ID NO: 220) are connected to form a light chain. In at least one embodiment, preferably, the heavy chain of the humanized anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 73; the light chain of the anti-TIGIT monoclonal antibody includes the amino acid sequence shown in SEQ ID NO: 90. In at least one embodiment, the humanized anti-TIGIT antibody is 6A10-5: H3E + L3A.

[0477] In some preferred embodiments of the present invention, the murine anti-TIGIT monoclonal antibody is humanized to obtain humanized anti-TIGIT antibodies such as BT-25H8-41, BT-7F4-12, BT-7F4-14, BT-6A10-31, BT-6A10-32 and BT-6A10-36, which change the heavy chain non-CDR1 region, such as HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3 and LFR4, but retain the binding activity to the human TIGIT antigen, and compared with the murine anti-TIGIT monoclonal antibodies BT-25H8, BT-7F4 and BT-6A10, the binding activity of the obtained humanized anti-TIGIT antibody to the human TIGIT antigen is not significantly decreased. Exemplarily, the exemplary humanized TIGIT antibody light and heavy chain combinations of the present invention are shown in Table 6.

[0478] Table 6 Examples of combinations of light and heavy chains of humanized TIGIT antibodies of the present invention

[0479]

[0480] Typically, the TIGIG antibody or antigen-binding fragment thereof obtained in the present invention is less than about 10 -6 M, for example, less than about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (K D )Combined with TIGIT.

[0481] In the present invention, the antibodies or antigen-binding fragments thereof provided can specifically recognize TIGIT from various genera, such as humans, mice, and monkeys (such as cynomolgus monkeys).

[0482] On the one hand, the present application provides an isolated TIGIT antigen binding protein, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), which may comprise at least one CDR in the antibody heavy chain variable region VH.

[0483] In some embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown in Table 1: For example, the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 and 27 and SEQ ID NOs: 29-42. In the present application, the VHCDR of the isolated antigen-binding protein may be divided in any form, as long as VH is identical to the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 and 27 and SEQ ID NOs: 29-42, the HCDR obtained by dividing in any form may fall within the protection scope of the present application.

[0484] For example, the VL may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 24, 26 and 28 and SEQ ID NOs: 43-56. In the present application, the VLCDR of the isolated antigen-binding protein may be divided in any form, as long as VL is identical to the amino acid sequence as shown in any one of SEQ ID NOs: 24, 26 and 29 and SEQ ID NOs: 43-56, the LCDR obtained by dividing in any form may fall within the protection scope of the present application.

[0485] The CDR of an antibody is also called the complementary determining region, which is a part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. The antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat / Chothia, etc. These coding systems are known in the art, and specific references can be made to, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In the present application, the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR being substituted, deleted and / or added with one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologues thereof are also contemplated, and the homologues may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR. In certain embodiments, the isolated antigen-binding proteins described herein are defined by the Kabat coding system.

[0486] In the present application, the antigen binding protein of the isolated anti-TIGIT antibody can bind to human TIGIT.

[0487] In the present application, the isolated antigen binding protein can bind to the extracellular part of human TIGIT.

[0488] In the present application, the antigen binding protein may comprise a heavy chain variable region (VH) and a light chain variable region (VL).

[0489] The VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.

[0490] In the present application, the HCDR1 of the antigen binding protein may comprise an amino acid sequence shown in any one of SEQ ID NOs: 1, 7, and 13. For example, the HCDR1 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0491] In the present application, the HCDR2 of the antigen binding protein may comprise an amino acid sequence shown in any one of SEQ ID NOs: 2, 8, and 14. For example, the HCDR2 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0492] In the present application, the HCDR3 of the antigen binding protein may comprise an amino acid sequence shown in any one of SEQ ID NOs: 3, 9, and 15. For example, the HCDR3 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0493] For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 2; and the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 3. For example, the antigen binding protein may comprise the chimeric antibody BT-25H8(464)-CHI, or, BT-25H8-1: H4A+L1A (25H8-41), BT-5H8-2: H4B+L2A (25H8-42), BT-25H8-3: H4C+L3A (25H8-43), BT-25H8-4: H4D+L6A (25H8-46) or BT-25H8-5: H2A+L1B (25H8-21), or a humanized antibody having the same HCDR3 (e.g., having HCDR1-3) therewith.

[0494] For example, the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 8; and the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9. For example, the antigen-binding protein may include the antibody BT-7F4-CHI or 7F4-1: H1A + L2A (7F4-12), 7F4-2: H1A + L4A (7F4-14, 7F4-3: H1B + L1A (7F4-31), 7F4-4: H7A + L2B (7F4-72), 7F4-5: H3A + L2A (7F4-32), or an antibody having the same HCDR3 therewith (e.g., having HCDR1-3 therewith).

[0495] For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 14; and the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the antigen binding protein may include antibodies BT-6A10-CHI to 6A10-1: H3A+L1A (6A104-31), 6A10-2: H3B+L2A (6A104-32), 6A10-3: H3C+L4A (6A104-34), 6A10-4: H3D+L6A (6A104-36), 6A10-5: H3E+L3A (6A104-33), or an antibody having the same HCDR3 therewith (e.g., having HCDR1-3 therewith).

[0496] The VL may include at least one, two or three of LCDR1, LCDR2 and LCDR3.

[0497] In the present application, the LCDR1 of the antigen binding protein may comprise an amino acid sequence shown in any one of SEQ ID NOs: 4, 10, and 16. For example, the LCDR1 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0498] In the present application, the LCDR2 of the antigen binding protein may comprise an amino acid sequence shown in any one of SEQ ID NOs: 5, 11, and 17. For example, the LCDR2 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0499] In the present application, the LCDR3 of the antigen binding protein may comprise an amino acid sequence shown in any one of SEQ ID NOs: 6, 12, and 18. For example, the LCDR3 sequence of the antigen binding protein may be defined according to the Kabat coding system.

[0500] For example, the LCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the antigen binding protein may comprise the chimeric antibody BT-25H8(464)-CHI, or, BT-25H8-1: H4A+L1A (25H8-41), BT-5H8-2: H4B+L2A (25H8-42), BT-25H8-3: H4C+L3A (25H8-43), BT-25H8-4: H4D+L6A (25H8-46) or BT-25H8-5: H2A+L1B (25H8-21) or a humanized antibody having the same LCDR3 (e.g., having LCDR1-3) therewith.

[0501] For example, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 10; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11; and the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the antigen binding protein may include the antibody BT-7F4-CHI or 7F4-1: H1A + L2A (7F4-12), 7F4-2: H1A + L4A (7F4-14, 7F4-3: H1B + L1A (7F4-31), 7F4-4: H7A + L2B (7F4-72), 7F4-5: H3A + L2A (7F4-32), or an antibody having the same LCDR3 therewith (e.g., having LCDR1-3 therewith).

[0502] For example, the VLCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 16; the LCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 17; and the VLCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the antigen binding protein may include antibodies BT-6A10-CHI to 6A10-1: H3A + L1A (6A104-31), 6A10-2: H3B + L2A (6A104-32), 6A10-3: H3C + L4A (6A104-34), 6A10-4: H3D + L6A (6A104-36), 6A10-5: H3E + L3A (6A104-33), or an antibody having the same VLCDR3 therewith (e.g., having VLCDR1-3 therewith).

[0503] In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.

[0504] For example, the antigen binding protein may comprise HCDR1-3 of the heavy chain variable region shown in any one of SEQ ID NOs: 23, 25 or 27. The antigen binding protein may comprise LCDR1-3 of the light chain variable region shown in any one of SEQ ID NOs: 24, 26 or 28.

[0505] In an exemplary embodiment, the H-FR1 may include the amino acid sequence shown in SEQ ID NOs:91-94; the H-FR2 may include the amino acid sequence shown in SEQ ID NOs:105-108; the H-FR3 may include the amino acid sequence shown in SEQ ID NOs:114-117; the H-FR4 may include the amino acid sequence shown in SEQ ID NOs:125-128; the L-FR1 may include the amino acid sequence shown in SEQ ID NOs:134-137; the L-FR2 may include the amino acid sequence shown in SEQ ID NOs:149-153; the L-FR3 may include the amino acid sequence shown in SEQ ID NOs:164-168; and the L-FR4 may include the amino acid sequence shown in SEQ ID NOs:179-182.

[0506] In the present application, the antigen binding protein may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region may comprise HCDR1-3 and H-FR1-4, and the light chain variable region may comprise LCDR1-3 and L-FR1-4.

[0507] For example, the antigen binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 23. For example, the antigen binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 24. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 91; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 105; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 114; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 125; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 136; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 151; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 181. For example, the antigen binding protein may include the antibody BT-25H8-CHI or an antigen binding protein having the same light chain variable region therewith. In the present application, the antigen binding protein includes a heavy chain (HC) and a light chain (LC), and the heavy chain of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 57. For example, the antigen binding protein may include the antibody BT-25H8-CHI or an antigen binding protein having the same heavy chain therewith. For example, the light chain of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO: 74. For example, the antigen-binding protein may include antibody BT-25H8-CHI or an antigen-binding protein having the same light chain therewith.

[0508] For example, the antigen binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 29. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 43. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:92; the H-FR2 may include the amino acid sequence shown in SEQ ID NO:106; the H-FR3 may include the amino acid sequence shown in SEQ ID NO:115; the H-FR4 may include the amino acid sequence shown in SEQ ID NO:126; the L-FR1 may include the amino acid sequence shown in SEQ ID NO:137; the L-FR2 may include the amino acid sequence shown in SEQ ID NO:152; the L-FR3 may include the amino acid sequence shown in SEQ ID NO:166; and the L-FR4 may include the amino acid sequence shown in SEQ ID NO:182.

[0509] In at least one specific embodiment, the heavy chain of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 58. For example, the light chain of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 75. For example, the antigen-binding protein may include antibody 25H8-1: H4A+L1A or an antigen-binding protein having the same light chain therewith.

[0510] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 30. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 44. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 93; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 107; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 116; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 127; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 138; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 153; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 168; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 183. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 30. For example, the antigen binding protein may include an antigen binding protein with the same heavy chain variable region as the antigen binding fragment 25H8-2: H4B + L2A. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 44. For example, the antigen-binding protein may include antibody 25H8-2:H4B+L2A or an antigen-binding protein having the same light chain variable region therewith.

[0511] In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 59. For example, the antigen binding protein may include antibody 25H8-2: H4B + L2A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 76. For example, the antigen binding protein may include antibody 25H8-2: H4B + L2A or an antigen binding protein having the same light chain.

[0512] In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 31. For example, the antigen binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 45. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:93; the H-FR2 may include the amino acid sequence shown in SEQ ID NO:107; the H-FR3 may include the amino acid sequence shown in SEQ ID NO:116; the H-FR4 may include the amino acid sequence shown in SEQ ID NO:126; the L-FR1 may include the amino acid sequence shown in SEQ ID NO:139; the L-FR2 may include the amino acid sequence shown in SEQ ID NO:154; the L-FR3 may include the amino acid sequence shown in SEQ ID NO:169; the L-FR4 may include the amino acid sequence shown in SEQ ID NO:183. For example, the heavy chain variable region of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:31. For example, the light chain variable region of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:45. For example, the antigen-binding protein may include antibody 25H8-3:H4C+L3A or an antigen-binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 60. For example, the antigen binding protein may include antibody 25H8-3: H4C + L3A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 77. For example, the antigen binding protein may include antibody 25H8-3: H4C + L3A or an antigen binding protein having the same light chain.

[0513] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 32. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 46. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:93; the H-FR2 may include the amino acid sequence shown in SEQ ID NO:107; the H-FR3 may include the amino acid sequence shown in SEQ ID NO:116; the H-FR4 may include the amino acid sequence shown in SEQ ID NO:128; the L-FR1 may include the amino acid sequence shown in SEQ ID NO:140; the L-FR2 may include the amino acid sequence shown in SEQ ID NO:155; the L-FR3 may include the amino acid sequence shown in SEQ ID NO:70; the L-FR4 may include the amino acid sequence shown in SEQ ID NO:183. For example, the heavy chain variable region of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:32. For example, the light chain variable region of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:46. For example, the antigen-binding protein may include antibody 25H8-4:H4D+L6 or an antigen-binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 61. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 78. For example, the antigen binding protein may include antibody 25H8-4:H4D+L6 or an antigen binding protein having the same light chain therewith.

[0514] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 33. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 47. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO:94; the H-FR2 may include the amino acid sequence shown in SEQ ID NO:108; the H-FR3 may include the amino acid sequence shown in SEQ ID NO:117; the H-FR4 may include the amino acid sequence shown in SEQ ID NO:128; the L-FR1 may include the amino acid sequence shown in SEQ ID NO:137; the L-FR2 may include the amino acid sequence shown in SEQ ID NO:152; the L-FR3 may include the amino acid sequence shown in SEQ ID NO:167; the L-FR4 may include the amino acid sequence shown in SEQ ID NO:184. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO:33. For example, the antigen binding protein may include an antigen binding protein with the same heavy chain variable region as the antigen binding fragment 25H8-5:H2A+L1B. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO:47. For example, the antigen binding protein may include antibody 25H8-5: H2A + L1B or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 62. For example, the antigen binding protein may include antibody 25H8-5: H2A + L1B or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 79. For example, the antigen binding protein may include antibody 25H8-5: H2A + L1B or an antigen binding protein having the same light chain.

[0515] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 25. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 26. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 95; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 109; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 118; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 129; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 141; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 156; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 171; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 181. In the present application, for example, the antigen-binding protein includes a heavy chain and a light chain, and the heavy chain of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 63. For example, the antigen-binding protein may include the antibody BT-7F4-CHI or an antigen-binding protein having the same heavy chain therewith. For example, the light chain of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO: 80. For example, the antigen binding protein may include antibody BT-7F4-CHI or an antigen binding protein having the same light chain therewith.

[0516] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 34. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 48. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 96; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 110; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 119; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 130; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 142; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 157; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 172; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 184. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 34. For example, the antigen binding protein may include the antigen binding fragment 7F4-1: H1A + L2A or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 48. For example, the antigen binding protein may include antibody 7F4-1: H1A + L2A or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 64. For example, the antigen binding protein may include antibody 7F4-1: H1A + L2A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 81. For example, the antigen binding protein may include antibody 7F4-1: H1A + L2A or an antigen binding protein having the same light chain.

[0517] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 34. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 49. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 96; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 110; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 119; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 130; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 143; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 158; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 173; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 153. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 34. For example, the antigen binding protein may include the antigen binding fragment 7F4-2: H1A + L4A or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 49. For example, the antigen binding protein may include antibody 7F4-2: H1A + L4A or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 64. For example, the antigen binding protein may include antibody 7F4-2: H1A + L4A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 82. For example, the antigen binding protein may include antibody 7F4-2: H1A + L4A or an antigen binding protein having the same light chain.

[0518] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 35. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 50. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 96; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 110; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 119; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 131; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 144; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 159; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 174; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 185. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 34. For example, the antigen binding protein may include the antigen binding fragment 7F4-3: H1B + L1A or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 48. For example, the antigen binding protein may include antibody 7F4-3: H1B + L1A or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 65. For example, the antigen binding protein may include antibody 7F4-3: H1B + L1A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 83. For example, the antigen binding protein may include antibody 7F4-3: H1B + L1A or an antigen binding protein having the same light chain.

[0519] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 36. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 51. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 97; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 110; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 120; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 131; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 142; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 157; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 172; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 182. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 34. For example, the antigen binding protein may include the antigen binding fragment 7F4-4: H7A + L2B or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 49. For example, the antigen binding protein may include antibody 7F4-4: H7A + L2B or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 66. For example, the antigen binding protein may include antibody 7F4-4: H7A + L2B or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 84. For example, the antigen binding protein may include antibody 7F4-4: H7A + L2B or an antigen binding protein having the same light chain.

[0520] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 37. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 48. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 98; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 111; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 121; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 130; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 142; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 157; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 172; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 182. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 37. For example, the antigen binding protein may include the antigen binding fragment 7F4-5: H3A + L2A or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 48. For example, the antigen binding protein may include antibody 7F4-5: H3A + L2A or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 67. For example, the antigen binding protein may include antibody 7F4-5: H3A + L2A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 81. For example, the antigen binding protein may include antibody 7F4-5: H3A + L2A or an antigen binding protein having the same light chain.

[0521] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 27. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 28. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 14; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 15; the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 16; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 99; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 112; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 122; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 132; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 145; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 160; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 175; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 181. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 27. For example, the antigen binding protein may include an antigen binding protein of the same heavy chain variable region as the antigen binding fragment BT-6A10-CHI. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 28.

[0522] For example, the antigen binding protein may include antibody BT-6A10-CHI or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:68. For example, the antigen binding protein may include antibody BT-6A10-CHI or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:85. For example, the antigen binding protein may include antibody BT-6A10-CHI or an antigen binding protein having the same light chain.

[0523] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 38. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 52. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 14; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 15; the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 16; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 100; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 123; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 133; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 146; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 161; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 176; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 182. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 38. For example, the antigen binding protein may include an antigen binding protein with the same heavy chain variable region as the antigen binding fragment 6A10-1: H3A + L1A. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 52. For example, the antigen-binding protein may include antibody 6A10-1:H3A+L1A or an antigen-binding protein having the same light chain variable region therewith.

[0524] In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 69. For example, the antigen binding protein may comprise antibody 6A10-1: H3A + L1A or an antigen binding protein having the same heavy chain thereof. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 86. For example, the antigen binding protein may comprise antibody 6A10-1: H3A + L1A or an antigen binding protein having the same light chain thereof.

[0525] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 39. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 53. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: SEQ ID NO: 13; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 14; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 15; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 101; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 123; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 133; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 147; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 162; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 177; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 182. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 39. For example, the antigen binding protein may include an antigen binding protein with the same heavy chain variable region as the antigen binding fragment 6A10-2: H3B + L2A. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 53. For example, the antigen-binding protein may include antibody 6A10-2:H3B+L2A or an antigen-binding protein having the same light chain variable region therewith.

[0526] In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 70. For example, the antigen binding protein may comprise antibody 6A10-2: H3B + L2A or an antigen binding protein having the same heavy chain thereof. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 87. For example, the antigen binding protein may comprise antibody 6A10-2: H3B + L2A or an antigen binding protein having the same light chain thereof.

[0527] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 40. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO: 54. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 15; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 15; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 17; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 102; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 123; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 133; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 148; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 163; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 186; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 182. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 40. For example, the antigen binding protein may include an antigen binding protein with the same heavy chain variable region as the antigen binding fragment 6A10-3: H3C + L4A. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 54. For example, the antigen-binding protein may include antibody 6A10-3:H3C+L4A or an antigen-binding protein having the same light chain variable region therewith.

[0528] In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 71. For example, the antigen binding protein may include antibody 6A10-3: H3C + L4A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 88. For example, the antigen binding protein may include antibody 6A10-3: H3C + L4A or an antigen binding protein having the same light chain.

[0529] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO:41. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO:55. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 103; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 123; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 135; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 149; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 164; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 179; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 182. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 41. For example, the antigen binding protein may include the antigen binding fragment 6A10-4: H3D + L6A or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 55. For example, the antigen binding protein may include antibody 6A10-4: H3D + L6A or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 72. For example, the antigen binding protein may include antibody 6A10-4: H3D + L6A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 89. For example, the antigen binding protein may include antibody 6A10-4: H3D + L6A or an antigen binding protein having the same light chain.

[0530] In the present application, the antigen-binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. For example, the antigen-binding protein may comprise HCDR1-3 of the heavy chain variable region shown in SEQ ID NO:42. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the antigen-binding protein may comprise LCDR1-3 of the light chain variable region shown in SEQ ID NO:56. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:8; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9; the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:10; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:18. For example, the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 104; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 113; the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 124; the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 133; the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 150; the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 165; the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 180; the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 142. For example, the heavy chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 42. For example, the antigen binding protein may include the antigen binding fragment 6A10-5: H3E + L3A or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 56. For example, the antigen binding protein may include antibody 6A10-5: H3E + L3A or an antigen binding protein having the same light chain variable region. In the present application, the antigen binding protein comprises a heavy chain and a light chain, and the heavy chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 73. For example, the antigen binding protein may include antibody 6A10-5: H3E + L3A or an antigen binding protein having the same heavy chain. For example, the light chain of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 90. For example, the antigen binding protein may include antibody 6A10-5: H3E + L3A or an antigen binding protein having the same light chain.

[0531] The humanized anti-TIGIT monoclonal antibody can also be obtained by affinity modification.

[0532] In some embodiments, the affinity maturation can be performed by optimizing the heavy chain CDR and light chain CDR of the antibody without changing the heavy chain framework region and the light chain framework region, and chimeric antibodies and humanized anti-TIGIT monoclonal antibody variants can be further obtained with high affinity to the TIGIT antigen.

[0533] In the present invention, the humanized anti-TIGIT monoclonal antibody variant has specific binding activity with the TIGIT antigen, can specifically recognize and bind to the TIGIT antigen protein expressed on cells in the body including tumor cells, and relieve the immunosuppressive effect of TIGIT on the body.

[0534] In one embodiment, the anti-TIGIT antibody is a chimeric antibody, wherein the heavy chain and light chain sequences further comprise human IgG4 heavy chain constant region and human kappa light chain constant region sequences to generate full-length heavy chain and light chain sequences.

[0535] In one embodiment, the full-length heavy chain sequence of the TIGIT antibody chimeric antibody of the present invention is:

[0536] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIMYSGYTSYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCARIYWWGRDYWGQGTT LTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 57). (In embodiments, depending on the expression system, the heavy chain may include a terminal K (lysine) residue).

[0537] In one embodiment, the full-length light chain sequence of the TIGIT antibody chimeric antibody of the present invention is:

[0538] DIQMTQSPSSLSASLGERVSLTCRASQDIGSTLTWLQQEPDGTIKRLIYATSSLEIGVPKRFSGSRTGSDYTLTISSLESEDFVDYYCLQYAISPYTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:74).

[0539] This antibody is hereinafter referred to as BT-25H8-CHI.

[0540] In one specific embodiment, the full-length heavy chain sequence of the TIGIT antibody chimeric antibody of the present invention is:

[0541] Heavy Chain:

[0542] QVQLRQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGTGGTNYNEKFKGKTTLTADKSSSTAYMQLSSLTSDDSAVYFCASKEDVYSWFAFWGQG TLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 63). In an embodiment, depending on the expression system, the heavy chain may include a terminal K (lysine) residue;

[0543] The light chain sequence is:

[0544] DIVMTQSHKFMSTSVGDRVRITCKASQDVKTTVAWFQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSIPWTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:80).

[0545] This antibody is hereinafter referred to as BT-7F4-CHI, or simply BT-7F4-CHI.

[0546] In one embodiment, the anti-TIGIT antibody is a chimeric antibody, and the heavy and light chain sequences further comprise a human IgG4 constant region sequence and a human kappa light chain constant region to produce full-length heavy and light chain sequences as described below:

[0547] Heavy Chain:

[0548] EVMLVESGGGLVKPGGSLKLSCAASGFTFSTYVMSWVRQTPEKRLEWVATMTSGGSHTYYSDNVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCAKWQISYYAMDYWGQG TSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 68). In one embodiment, depending on the expression system, the heavy chain may include a terminal K (lysine) residue;

[0549] The light chain sequence is:

[0550] DIQMNQSPSSLSASLGDTITITCHASQNINVWLNWYQQKPGNIPKLLIYKASTLHTGVPSRFSGRGSGTGFTLSISSLQPEDIATYYCQQGQTYPYTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:85).

[0551] This antibody is hereinafter referred to as BT-6A10-CHI, or simply BT-6A10-CHI. All of the above specific (ie, excluding variables) sequences are sequences of the antibody BT-6A10-CHI.

[0552] In one embodiment, the anti-TIGIT antibody is a fully human antibody, and the heavy and light chains further comprise a human IgG4 constant region sequence and a human kappa chain light chain constant region, respectively, to produce full-length heavy and light chain sequences as described below.

[0553] In a specific embodiment, the heavy chain of the fully human TIGIT antibody of the present invention is:

[0554] QVQLQESGPGLVKPSGTLSLTCAVTGYSITSDYAWNWVRQPPGKGLEWMGYIMYSGYTSYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARIYWWGRDYWGRGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 58), in an embodiment, depending on the expression system, the heavy chain may include a terminal K (lysine) residue.

[0555] In a specific embodiment, the light chain sequence of the fully human TIGIT antibody of the present invention is:

[0556] DIQMTQSPSSSLSASVGDRVTITCRASQDIGSTLTWYQQKPEKAPKRLIYATSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYAISPYTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:75).

[0557] This antibody is referred to hereinafter as BT-25H8-41. All specific (ie excluding variables) sequences described above are the sequences of antibody 25H8-1:H4A+L1A (25H8-41).

[0558] In a specific embodiment, the anti-TIGIT antibody of the present invention is a fully human IgG1 antibody, and the heavy chain and light chain sequences further comprise a human IgG4 heavy chain constant region sequence and a human κ chain light chain constant region sequence to produce the full-length heavy chain and light chain sequences as described below (SEQ ID NO: 59 and SEQ ID NO: 76):

[0559] Heavy chain

[0560] QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYIMYSGYTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIYWWGRDYWGQGTMVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV

[0561] FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:59);

[0562] Light chain

[0563] DIVMTQTPLSLSVTPGQPASISCRASQDIGSTLTWYLQKPGQSPQRLIYATSSLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQYAISPYTFGPGTKVDIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:76)

[0564] This antibody is hereinafter referred to as 25H8-2:H4B+L2A, or BT-25H8-42 for short. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-25H8-42.

[0565] In a very specific aspect, the anti-TIGIT antibody of the present invention is a fully human IgG1 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region and a human κ-type light chain constant region u to produce the full-length heavy chain and light chain sequences as described below: (SEQ ID NO: 60 and SEQ ID NO: 77):

[0566] QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYIMYSGYTSYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARIYWWGRDYWGRGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:60);

[0567] EIVMTQSPATLSVSPGERATLSCRASQDIGSTLTWYQQKPGQAPRRLIYATSSLETGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQYAISPYTFGPGTKVDIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:77);

[0568] This antibody is hereinafter referred to as BT-25H8-3:H4C+L3A, or simply BT-25H8-43. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-25H8-43.

[0569] In a very specific aspect, the anti-TIGIT antibody of the present invention is a fully human IgG1 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region and a human κ-type light chain constant region to produce the full-length heavy chain and light chain sequences as described below: to produce the full-length heavy chain and light chain sequences as described below: (SEQ ID NO: 61 and SEQ ID NO: 78):

[0570] QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYIMYSGYTSYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARIYWWGRDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:61);

[0571] EIVLTQSPDFQSVTPKEKVTITCRASQDIGSTLTWYQQKPDQSPKRLIYATSSLESGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCLQYAISPYTFGPGTKVDIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:78);

[0572] This antibody is hereinafter referred to as BT-25H8-4:H4D+L6A, or simply 25H8-46. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-25H8-46.

[0573] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy and light chain variable region sequences further comprise human constant region sequences to produce full length heavy and light chain sequences as described below: (SEQ ID NO:62 and SEQ ID NO:79):

[0574] QITLKESGPTLVKPTQTLTLTCTVTGYSITSDYAWNWIRQPPGKALEWMGYIMYSGYTSYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARIYWWGRDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:62);

[0575] DIQMTQSPSSSLSASVGDRVTITCRASQDIGSTLTWYQQKPEKAPKRLIYATSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYAISPYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:79);

[0576] This antibody is hereinafter referred to as BT 25H8-5:H2A+L1B, or simply BT-25H8-21. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-25H8-21.

[0577] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:64 and SEQ ID NO:81):

[0578] QVQLVQSGAEVKKPGSSVKVSCKASGYAFTNYLIEWVRQAPGQGLEWIGVINPGTGGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASKEDVYSWFAFWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:64);

[0579] DIVMTQTPLSLSVTPGQPASISCKASQDVKTTVAWYLQKPGQPPQLLIYWASTRHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSIPWTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 81);

[0580] This antibody is hereinafter referred to as 7F4-1:H1A+L2A, or BT-7F4-12 for short. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-7F4-12.

[0581] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:64 and SEQ ID NO:82):

[0582] QVQLVQSGAEVKKPGSSVKVSCKASGYAFTNYLIEWVRQAPGQGLEWIGVINPGTGGTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASKEDVYSWFAFWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:64);

[0583] DIVMTQSPDSLAVSLGERATINCKASQDVKTTVAWYQQKPGQPPKLLIYWASTRHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSIPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:82);

[0584] This antibody is hereinafter referred to as BT-7F4-2:H1A+L4A, or simply BT-7F4-14. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-7F4-14.

[0585] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region, to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:65 and SEQ ID NO:83):

[0586] QVQLVQSGSELKKPGASVKVSCKASGYAFTNYLIEWVRQAPGQGLEWIGVINPGTGGTNYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCASKEDVYSWFAFWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 65);

[0587] DIQMTQSPSSLSASVGDRVTITCKASQDVKTTVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSIPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83) This antibody is hereinafter referred to as BT-7F4-3:H1B+L1A, or simply BT-7F4-11. All of the above specific (ie, excluding variables) sequences are the sequences of antibody BT-7F4-13.

[0588] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:66 and SEQ ID NO:84):

[0589] QVQLVESGGGVVQPGGSLRLSCAASGYAFTNYLIEWVRQAPGKGLEWIGVINPGTGGTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASKEDVYSWFAFWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:66);

[0590] DIVMTQTPLSLSVTPGQPASISCKASQDVKTTVAWYLQKPGQPPQLLIYWASTRHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSIPWTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:84);

[0591] This antibody is hereinafter referred to as 7F4-4:H7A+L2B, or BT-7F4-72 for short. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-7F4-72.

[0592] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region, to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:67 and SEQ ID NO:81):

[0593] QVQLVESGGGVVQPGGSLRLSCAASGYAFTNYLIEWVRQAPGKGLEWIGVINPGTGGTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASKEDVYSWFAFWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:67);

[0594] DIVMTQTPLSLSVTPGQPASISCKASQDVKTTVAWYLQKPGQPPQLLIYWASTRHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSIPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 81) This antibody is hereinafter referred to as BT-7F4-5:H3A+L2A, or simply BT-7F4-32. All of the above specific (ie, excluding variables) sequences are the sequences of antibody BT-7F4-32.

[0595] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:69 and SEQ ID NO:86):

[0596] QVQLVESGGGVVQPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGLEWVATMTSGGSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWQISYYAMDYWGRG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:69);

[0597] DIQLTQSPSFLSASVGDRVTITCHASQNINVWLNWYQQKPGKAPKLLIYKASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQGQTYPYTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:86);

[0598] This antibody is hereinafter referred to as BT-6A10-1:H3A+L1A, or simply BT-6A10-31. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-6A10-31.

[0599] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region, to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:70 and SEQ ID NO:87):

[0600] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGLEWVATMTSGGSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWQISYYAMDYWGRG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:70);

[0601] DIVMTQTPLSLSVTPGQPASISCHASQNINVWLNWYLQKPGQSPQLLIYKASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQGQTYPYTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:87).

[0602] This antibody is hereinafter referred to as BT-6A10-2:H3B+L2A, or simply BT-6A10-32. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-6A10-32.

[0603] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:71 and SEQ ID NO:88):

[0604] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGLEWVATMTSGGSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWQISYYAMDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:71);

[0605] DIVMTQSPDSLAVSLGERATINCHASQNINVWLNWYQQKPGQPPKLLIYKASTLHSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGQTYPYTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:88).

[0606] This antibody is hereinafter referred to as 6A10-3:H3C+L4A, or BT-6A10-34 for short. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-6A10-34.

[0607] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region, to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:72 and SEQ ID NO:89):

[0608] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGLEWVATMTSGGSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWQISYYAMDYWGRG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:70);

[0609] DIVMTQTPLSLSVTPGQPASISCHASQNINVWLNWYLQKPGQSPQLLIYKASTLHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQGQTYPYTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:87).

[0610] This antibody is hereinafter referred to as 6A10-4:H3D+L6A, or simply BT-6A10-36. All of the above specific (ie excluding variables) sequences are sequences of antibody BT-6A10-36.

[0611] In a very specific aspect, the anti-TIGIT antibody is a fully human IgG4 antibody, and the heavy chain and light chain variable region sequences further comprise a human IgG4 heavy chain constant region sequence and a human kappa type light chain constant region to produce the full length heavy chain and light chain sequences as described below: (SEQ ID NO:73 and SEQ ID NO:90):

[0612] EVQLVESGGGLVQPGGSLRLSCSASGFTFSTYVMSWVRQAPGKGLEWVATMTSGGSHTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAKWQISYYAMDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:73);

[0613] EIVMTQSPATLSLSPGERATLSCHASQNINVWLNWYQQKPGQAPRLLIYKASTLHTGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQGQTYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:90);

[0614] The antibody is hereinafter referred to as 6A10-5:H3E+L3A, or simply as BT-6A10-33. All of the above specific (i.e., excluding the variable) sequences are the sequences of antibody BT-6A10-33.

[0615] In other very specific aspects, the present invention provides anti-TIGIT antibodies respectively referred to as BT-25H8-CH1, BT-7F4-CHI, BT-6A10-CHI, BT-25H8-41, BT-25H8-42, BT-25H8-43, BT-25H8-46, BT-25H8-21, BT-7F4-12, BT-7F4-14, BT-7F4-11, BT-7F4-72, BT-7F4-32, BT-6A10-31, BT-6A10-32, BT-6A10-34 and BT-6A10-36, BT-6A10-33.

[0616] "Humanized antibody" is an antibody that retains the reactivity of non-human antibodies while having lower immunogenicity in humans. For example, it can be obtained by retaining the non-human antibody CDR region and replacing the rest of the antibody with the human antibody counterpart (i.e., the constant region and the framework portion of the variable region).

[0617] An important advantage of the antibody or its antigen-binding fragment is that it has high in vitro binding activity and cross-species binding activity with TIGIT.

[0618] An important advantage of the antibody or its antigen-binding fragment is that it has the activity of blocking the binding of PVR to TIGIT.

[0619] An important advantage of the antibody or antigen-binding fragment thereof is that it has a significant anti-tumor effect.

[0620] An important advantage of the antibody or antigen-binding fragment thereof is that it can reduce the proportion of Treg cells.

[0621] Due to having one or more of the above characteristics, the antibody or antigen-binding fragment thereof that specifically binds to TIGIT of the present invention can be used as an antibody drug.

[0622] Coding nucleic acid

[0623] In one aspect, the present application provides nucleic acid molecules 1 and 2 encoding the heavy chain variable region (VH) / light chain variable region (VL) of the antibody or antigen binding portion thereof that specifically binds to TIGIT. The variable region encoding nucleic acids of exemplary anti-TIGIT monoclonal antibodies of the present invention are shown in Table 7 in the Examples.

[0624] Table 7

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632] In one embodiment, the TIGIT monoclonal antibody is a chimeric antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the TIGIT monoclonal antibody as a chimeric antibody has the nucleotide sequence shown in SEQ ID NO: 186 and SEQ ID NO: 203 in Table 7 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8(464)-CHI.

[0633] In one embodiment, the TIGIT monoclonal antibody is a chimeric antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the TIGIT monoclonal antibody as a chimeric antibody has the nucleotide sequence shown in SEQ ID NO: 192 and SEQ ID NO: 209 in Table 7 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 7F4(465)-CHI.

[0634] In one embodiment, the TIGIT monoclonal antibody is a chimeric antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the TIGIT monoclonal antibody as a chimeric antibody has the nucleotide sequence shown in SEQ ID NO: 197 and SEQ ID NO: 214 in Table 7 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 6A10(467)-CHI.

[0635] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 187 and SEQ ID NO: 204 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8-1: H4A + L1A.

[0636] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 188 and SEQ ID NO: 205 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8-2: H4B + L2A.

[0637] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 189 and SEQ ID NO: 206 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8-3: H4C + L3A.

[0638] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 190 and SEQ ID NO: 207 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8-3: H4C + L3A.

[0639] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 191 and SEQ ID NO: 208 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8-4: H4D + L6A.

[0640] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 188 and SEQ ID NO: 205 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 25H8-5: H2A + L1B.

[0641] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 193 and SEQ ID NO: 210 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 7F4-1: H1A + L2A.

[0642] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 193 and SEQ ID NO: 211 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 7F4-2: H1A + L4A.

[0643] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 194 and SEQ ID NO: 212 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 7F4-3: H1B + L1A.

[0644] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 195 and SEQ ID NO: 213 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 7F4-4: H7A + L2B.

[0645] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 196 and SEQ ID NO: 210 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 7F4-5: H3A + L2A.

[0646] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 198 and SEQ ID NO: 215 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody is 6A10-1: H3A + L1A.

[0647] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 199 and SEQ ID NO: 216 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody 6A10-2: H3B + L2A.

[0648] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 200 and SEQ ID NO: 217 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody 6A10-3: H3C + L4A.

[0649] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 201198 and SEQ ID NO: 218 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody 6A10-4: H3D + L6A.

[0650] In one embodiment, the TIGIT monoclonal antibody is a humanized antibody. In a specific embodiment, the isolated nucleic acid encoding the VH and VL of the humanized TIGIT monoclonal antibody has the nucleotide sequence shown in SEQ ID NO: 202 and SEQ ID NO: 219 in Table 8 of Example 4. In at least one exemplary embodiment, the TIGIT antibody 6A10-5: H3E + L3A.

[0651] Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the present application can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0652] The nucleic acid of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes), cDNAs encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be collected from gene banks.

[0653] Preferred nucleic acid molecules of the present application include those encoding the VH and VL sequences or CDRs of TIGIT monoclonal antibodies. Once the DNA fragments encoding VH and VL are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these operations, the DNA fragment encoding VH or VL is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" refers to two DNA fragments being linked together so that the amino acid sequences encoded by the two DNA fragments are in the reading frame.

[0654] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably connecting the VH encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). The sequence of the human heavy chain constant region gene is known in the art, and the DNA fragments including these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but preferably IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the DNA encoding the VH region can be operably connected to another DNA molecule encoding only the heavy chain CH1 constant region.

[0655] The isolated DNA encoding the VL region can be converted into a full-length light chain gene by operably connecting the VL encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequence of the human light chain constant region gene is known in the art, and the DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa and lambda constant region.

[0656] To create an scFv gene, the DNA fragments encoding VH and VL can be operably linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VH and VL regions are connected by the flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0657] Preparation of monoclonal antibodies in this application

[0658] The monoclonal antibodies of the present application can be prepared using the somatic cell hybridization (hybridoma) technology of Kohler and Milstein (1975) Nature 256: 495. Other embodiments for preparing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display technology. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

[0659] Preferably, the preparation of the mouse anti-TIGIT monoclonal antibody of the present application adopts somatic cell hybridization (hybridoma) technology

[0660] Preferably, the chimeric antibodies and humanized antibodies of the present application can also be prepared using, for example, recombinant DNA technology combined with gene transfection methods, and generated in host cell transfectomas (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors, so that the genes are operably linked to transcription and translation regulatory sequences. In this case, the term "operably linked" means that the antibody gene is linked to the vector, so that the transcription and translation control sequences in the vector exercise their established functions of regulating antibody gene transcription and translation.

[0661] The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, such as the adenovirus major late promoter (AdMLP) and polyoma virus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter may be used. In addition, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type I (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0662] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is inserted into the expression vector of the heavy chain constant region and the light chain constant region that have encoded the desired subtype and constructs the full-length antibody gene, so that VH is operably connected to the CH in the vector, and VL is operably connected to the CL in the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector, so that the signal peptide is connected to the amino terminus of the antibody chain gene in the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (that is, a signal peptide from a non-immunoglobulin).

[0663] In addition to the antibody chain gene and the regulatory sequence, the recombinant expression vector of the present application may carry other sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene can be used to select the host cell into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216; 4,634,665 and 5,179,017). For example, the selectable marker gene usually confers drug resistance, such as G418, hygromycin, or methotrexate resistance, to the host cell into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for methotrexate selection / amplification of dhfr host cells) and neo genes (for G418 selection).

[0664] For the expression of light and heavy chains, expression vectors encod...

Claims

1. An isolated antibody or antigen-binding fragment that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain complementary determining region (HCDRs) combination consisting of HCDR 1, HCDR 2 and HCDR3 and a light chain complementary determining region (LCDRs) combination consisting of LCDR 1, LCDR 2 and LCDR 3, wherein the antibody comprises one, two, or more of the following CDRs: (a) HCDR 1, which comprises the amino acid sequence shown in SEQ ID NO: 1, 7 or 13, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 1, 7 or 13; (b) HCDR 2, which comprises the amino acid sequence shown in SEQ ID NO: 2, 8 or 14, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 2, 8 or 14; (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, 9 or 15, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 3, 9 or 15; (d) LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, 10 or 16, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence shown in SEQ ID NO: 4, 10 or 16; (e) LCDR2, which comprises the amino acid sequence shown in SEQ ID NO 5, 11 or 17, or an amino acid sequence having at least 85% sequence homology to 5, 11 or 17; (f) LCDR 3, whose sequence comprises the amino acid sequence shown in SEQ ID NO: 6, 12 or 18; or an amino acid sequence having at least 90% sequence homology with formula 4 shown in SEQ ID NO: 6, 12 or 18.

2. An isolated antibody or antigen-binding fragment that specifically binds to TIGIT, comprising a heavy chain variable region (VH) H and a light chain variable region (VL), wherein: According to the IMGT numbering system, The heavy chain variable region of the anti-TIGIT antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1-3, respectively; and the light chain variable region of the anti-TIGIT antibody comprises LCDR1-LCDR3 as shown in SEQ ID NOs: 4-6, respectively, or The heavy chain variable region of the immunoglobulin comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 7, 8 and -9, respectively, and the light chain variable region of the anti-TIGIT antibody comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, or The heavy chain variable region of the anti-TIGIT antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 13, 14 and 15, respectively, and the light chain variable region of the anti-TIGIT antibody comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18.

3. The antibody or antigen-binding fragment of any one of claims 1-2, further comprising a heavy chain variable region framework sequence HC-FR1, HC-FR2, HC-FR3 and HC-FR4 located between the HCDRs, thereby forming a sequence of the formula: (HC-FR1)-(HCDR1)-(HC-FR2)-(HCDR2)-(HC-FR3)-(HCDR3)-(HC-FR4), or further comprising a variable region light chain framework sequence LC-FR1, LC-FR2, LC-FR3 and LC-FR4 located between the LCDRs, thereby forming a sequence of the formula: (LC-FR1)-(LCDR1)-(LCFR2)-(HVR-L2)-(LC-FR3)-(LCDR3)-(LC-FR4), or optional The heavy chain framework sequences HC-FR1, HC-FR2, HC-FR3 and HC-FR comprise one or more of the following HC-FRs: The HC-FR1 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 91-104, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 91-104; The HC-FR2 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 105-113, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 105-113 The HC-FR3 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 114-124, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 114-124 The HC-FR4 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 125-135, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 125-135; or Further optionally, the light chain further comprises variable region light chain framework sequences LC-FR1, LC-FR2, LC-FR3 and LC-FR4 located between the LCDRs, thereby forming a sequence of the following formula: (LC-FR1)-(LCDR1)-(LC-FR2)-(LCDR2)-(LC-FR3)-(LCDR3)-(LC-FR4), Wherein the light chain framework sequences LC-FR1, LC-FR2, LC-FR3 and LC-FR4 comprise one or more of the LC-FRs described below: The LC-FR1 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 136-150, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 136-150; The LC-FR2 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 151-165, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 151-165; The LC-FR3 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 166-180, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 166-180; The LC-FR4 sequence is an amino acid sequence as shown in any one of SEQ ID NOs: 181-185, or an amino acid sequence having at least 85% homology to an amino acid sequence as shown in any one of SEQ ID NOs: 181-185.

4. An isolated antibody or antigen-binding fragment that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 23, 25 or 27, or the amino acid sequence shown in any one of SEQ ID NOs: 29-42, or has a sequence homology of not less than 85% with the amino acid sequence shown in any one of SEQ ID NOs: 29-42, and / or The light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 26 or 28, or the amino acid sequence shown in any one of SEQ ID NOs: 43-56, or has a sequence homology of not less than 85% with the amino acid sequence shown in any one of SEQ ID NOs: 43-56.

5. The isolated antibody or antigen-binding portion thereof that specifically binds to TIGIT according to any one of claims 1 to 4, wherein the VH comprises an amino acid sequence selected from any one of SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27; or the VL comprises an amino acid sequence selected from any one of SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28; Or the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 29-33, or an amino acid sequence selected from any one of SEQ ID NOs: 34-37, or an amino acid sequence selected from any one of SEQ ID NOs: 38-42; the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 43-47, or an amino acid sequence selected from any one of SEQ ID NOs: 48-51, or an amino acid sequence selected from any one of SEQ ID NOs: 52-56; or in, The VH comprises the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises the amino acid sequence shown in SEQ ID NO: 24; or wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; or The VH of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises the amino acid sequence shown in SEQ ID NO: 28; or The VH of the anti-TIGIT antibody comprises the amino acid sequence shown in SEQ ID NO: 29, and the amino acid sequence of the anti-TIGIT antibody comprises the light chain variable region shown in SEQ ID NO: 43; or The VH comprises the amino acid sequence shown in SEQ ID NO:30, and the VL comprises the amino acid sequence shown in SEQ ID NO:44; or The VH comprises the amino acid sequence shown in SEQ ID NO:31, and the VL comprises the amino acid sequence shown in SEQ ID NO:45; or The VH comprises as shown in SEQ ID NO:32, and the amino acid sequence of the light chain variable region of the anti-TIGIT antibody is as shown in SEQ ID NO:46; or, The VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 47; or, The amino acid sequence of the VH is shown in SEQ ID NO: 34, and the amino acid sequence of the VL is shown in SEQ ID NO: 48 or 49; or, The amino acid sequence of the VH is shown in SEQ ID NO:35, and the amino acid sequence of the VL is shown in SEQ ID NO:50; or, The VH comprises the amino acid sequence shown in SEQ ID NO:36, and the VL comprises the amino acid sequence shown in SEQ ID NO:51; or, The VH comprises the amino acid sequence shown in SEQ ID NO:37, and the VL comprises the amino acid sequence shown in SEQ ID NO:48; or, The VH comprises the amino acid sequence shown in SEQ ID NO:38, and the VL comprises the amino acid sequence shown in SEQ ID NO:52; or, The VH comprises the amino acid sequence shown in SEQ ID NO:39, and the VL comprises the amino acid sequence shown in SEQ ID NO:53; or, The VH comprises the amino acid sequence shown in SEQ ID NO:40, and the VL comprises the amino acid sequence shown in SEQ ID NO:54; or, The VH comprises the amino acid sequence shown in SEQ ID NO:41, and the VL comprises the amino acid sequence shown in SEQ ID NO:55; or, The VH comprises the amino acid sequence shown in SEQ ID NO:42, and the VL comprises the amino acid sequence shown in SEQ ID NO:

56.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The anti-TIGIT antibody further comprises a heavy chain constant region and a light chain constant region, and the species origin of the heavy chain constant region and the light chain constant region is selected from any one of mouse and human; Or, the optional heavy chain constant region sequence is selected from human IgG1 or human IgG4; Or, optionally, the light chain constant region is a human kappa or lambda chain; Or optionally, the heavy chain constant region comprises or has an amino acid sequence as shown in SEQ ID NO:222, or comprises or has an amino acid sequence that has no less than 85% homology with the amino acid sequence shown in SEQ ID NO:222, and the light chain constant region comprises or has an amino acid sequence as shown in SEQ ID NO:223; or comprises or has an amino acid sequence that has no less than 85% homology with the amino acid sequence shown in SEQ ID NO:

223.

7. An isolated anti-TIGIT antibody or antigen-binding fragment thereof, which is a full-length antibody comprising a heavy chain (HC) and a light chain (LC), wherein: The HC comprises the amino acid sequence shown in any one of SEQ ID NOs: 57-73, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 57-73; or optionally, the LC comprises the amino acid sequence shown in any one of SEQ ID NOs: 74-90, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 74-90.

8. The antibody or antigen-binding fragment thereof according to claim 8, wherein the HC and LC comprise an amino acid sequence combination having at least 85% sequence identity with an amino acid sequence combination selected from any one of the following groups: a) HC: SEQ ID NO: 57 and LC: SEQ ID NO: 74; b) HC: SEQ ID NO: 58 and LC: SEQ ID NO: 75; c) HC: SEQ ID NO: 59 and LC: SEQ ID NO: 76; d) HC: SEQ ID NO: 60 and LC: SEQ ID NO: 77; e) HC: SEQ ID NO: 61 and LC: SEQ ID NO: 78; f) HC: SEQ ID NO: 62 and LC: SEQ ID NO: 79; g) HC: SEQ ID NO: 63 and LC: SEQ ID NO: 80; h) HC: SEQ ID NO: 64 and LC: SEQ ID NO: 81; i) HC: SEQ ID NO: 64 and LC: SEQ ID NO: 82; j) HC: SEQ ID NO: 65 and LC: SEQ ID NO: 83; k) HC: SEQ ID NO: 66 and LC: SEQ ID NO: 84; l) HC: SEQ ID NO: 67 and LC: SEQ ID NO: 81; o) HC: SEQ ID NO: 69 and LC: SEQ ID NO: 86; p) HC: SEQ ID NO:70 and LC: SEQ ID NO:87; q) HC: SEQ ID NO:71 and LC: SEQ ID NO:88; r) HC: SEQ ID NO: 72 and LC: SEQ ID NO: 89; and s) HC: SEQ ID NO:73 and LC: SEQ ID NO:

90.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antigen-binding fragment is one selected from Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFvdi-scFv or VHH and / or dAb, wherein the antibody is one selected from monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody and fully human antibody.

10. The isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-9, which (a) binds to human TIGIT; (b) binds to monkey TIGIT; (c) does not bind to mouse TIGIT; (d) blocks TIGIT-PVR interaction; (e) promotes T cell activation; (f) triggers ADCC of immune cells against TIGIT-positive cells; (g) has an anti-tumor effect in vivo; and / or (h) has a synergistic anti-tumor effect with PD L1 antibody.

11. An isolated antibody or antigen-binding fragment thereof that specifically binds to TIGIT, which cross-competes with the antibody of any one of claims 1-10 for binding to TIGIT.

12. A polypeptide comprising the isolated antibody or antigen-binding fragment thereof that specifically binds to TIGIT according to any one of claims 1 to 12.

13. An antibody-drug conjugate molecule comprising the isolated antigen-binding protein according to any one of claims 1 to 13 or the polypeptide according to claim 12.

14. An isolated nucleic acid molecule comprising: (1) a nucleotide sequence 1 encoding the VH of the TIGIT antibody or antigen-binding fragment of any one of claims 2 to 5, and (2) a nucleotide sequence 2 encoding the VL of the TIGIT antibody or antigen-binding fragment of any one of claims 2 to 5, wherein The nucleotide sequence 1 comprises a nucleotide sequence selected from any one of SEQ ID NOs: 186-202, or a nucleotide sequence having a sequence homology of not less than 85% with the nucleotide sequence shown in any one of SEQ ID NOs: 186-202; The nucleotide sequence comprises a nucleotide sequence selected from any one of SEQ ID NOs: 203-219, or a nucleotide sequence having a sequence homology of not less than 85% with the nucleotide sequence shown in any one of SEQ ID NOs: 203-219.

15. A vector comprising the isolated nucleic acid molecule of claim 15.

16. A cell comprising the isolated antibody or antigen-binding fragment of any one of claims 1-11, the polypeptide of claim 12, the immunoconjugate of claim 13, the isolated nucleic acid molecule of claim 14 and / or the vector of claim 15.

17. A method for preparing the isolated antigen-binding protein of any one of claims 1 to 11 or the polypeptide of claim 12, the method comprising: (1) preparing mouse anti-TIGIT antibodies by the hybridoma method; (2) culturing the cells of claim 16 under conditions suitable for expressing the isolated antigen-binding protein of any one of claims 1 to 11 or the polypeptide of claim 12.

18. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1 to 12, the polypeptide of claim 13, the immunoconjugate of claim 14, the isolated nucleic acid molecule of claim 15, the vector of claim 16, the cell of claim 17, and / or a pharmaceutically acceptable adjuvant and / or excipient.

19. The isolated antibody or antigen-binding fragment that specifically binds to TIGIT according to any one of claims 1 to 12, or the polypeptide according to claim 13, or the pharmaceutical combination according to claim 18, for preventing, alleviating and / or treating tumors, wherein: The tumor includes a solid tumor, or the tumor includes a tumor associated with the expression of a protein selected from the group consisting of TIGIT, CD155 and CD112.

20. Use of the isolated antigen-binding protein according to any one of claims 1 to 11, the polypeptide according to claim 12, the immunoconjugate according to claim 13, the isolated nucleic acid molecule according to claim 14, the vector according to claim 15, the cell according to claim 16, or the drug combination according to any one of claims 17 to 19 in the preparation of a medicament for regulating immune response or for preventing, alleviating and / or treating tumors; Optionally, the tumor includes a tumor associated with the expression of a protein selected from the group consisting of: TIGIT, CD155 and CD112.

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