Anti-TIGIT antibodies and uses thereof

Abstract

The present disclosure relates to anti-TIGIT antibodies or antigen binding fragments thereof, and antibody drug conjugates.

Description

[0001] Priority Statement

[0002] This application claims the benefit of PCT / CN2023 / 120398, filed on September 21, 2023, the entire text of which is incorporated herein by reference. Technical Field

[0003] This disclosure relates to anti-TIGIT antibodies or antigen-binding fragments thereof, and antibody-drug conjugates. Background Technology

[0004] Cancer is one of the leading causes of death worldwide. According to statistics from the World Health Organization, in 2018, there were 18.1 million new cancer cases and 9.6 million cancer deaths globally. In China, there were 3.8 million newly diagnosed cancer cases and 2.3 million cancer deaths.

[0005] The recent clinical and commercial successes of anticancer antibodies have sparked great interest in antibody-based therapeutics. There is a need to develop antibodies for various antibody-based therapies to treat cancer or autoimmune diseases. Summary of the Invention

[0006] This disclosure relates to anti-TIGIT antibodies, their antigen-binding fragments, and their uses.

[0007] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TIGIT (a T-cell immune receptor having Ig and ITIM domains), comprising: The heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains an amino acid sequence having at least 80% identity with the selected VH CDR1 amino acid sequence, the VH CDR2 region contains an amino acid sequence having at least 80% identity with the selected VH CDR2 amino acid sequence, and the VH CDR3 region contains an amino acid sequence having at least 80% identity with the selected VH CDR3 amino acid sequence; and The light chain variable region (VL) comprises CDR1, 2, and 3, wherein the VL CDR1 region contains an amino acid sequence having at least 80% identity with the selected VL CDR1 amino acid sequence, the VL CDR2 region contains an amino acid sequence having at least 80% identity with the selected VL CDR2 amino acid sequence, and the VL CDR3 region contains an amino acid sequence having at least 80% identity with the selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; (2) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, respectively; (3) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 61, and 15, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 62, and 15, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22-24, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 25-27, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28-30, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31-33, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34-36, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37-39, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (11) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 40-42, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22-24, respectively; and (12) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 43-45, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28-30.

[0008] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 1-3, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 4-6, respectively.

[0009] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 7-9, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 10-12, respectively.

[0010] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 13-15, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 16-18, respectively.

[0011] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 13, 61, and 15, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 16-18, respectively.

[0012] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 13, 62, and 15, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 16-18, respectively.

[0013] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 19-21, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 22-24, respectively.

[0014] In some embodiments, according to the Kabat definition, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 25-27, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 28-30, respectively.

[0015] In some embodiments, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 31-33, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 4-6, respectively.

[0016] In some embodiments, according to Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 34-36, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 10-12, respectively.

[0017] In some embodiments, according to Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 37-39, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 16-18, respectively.

[0018] In some embodiments, according to Chothia's definition, the VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 40-42, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 22-24, respectively.

[0019] In some embodiments, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 43-45, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 28-30, respectively.

[0020] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TIGIT, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) containing an amino acid sequence having at least 90% identity with a selected VH sequence; and a light chain variable region (VL) containing an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO: 47; (2) The selected VH sequence is SEQ ID NO: 48 or 65, and the selected VL sequence is SEQ ID NO: 49; (3) The selected VH sequence is SEQ ID NO: 50, and the selected VL sequence is SEQ ID NO: 51; (4) The selected VH sequence is SEQ ID NO: 63, and the selected VL sequence is SEQ ID NO: 51; (5) The selected VH sequence is SEQ ID NO: 64, and the selected VL sequence is SEQ ID NO: 51; (6) The selected VH sequence is SEQ ID NO: 52, and the selected VL sequence is SEQ ID NO: 53; and (7) The selected VH sequence is SEQ ID NO: 54, and the selected VL sequence is SEQ ID NO: 55.

[0021] In some embodiments, the VH comprises the sequence of SEQ ID NO: 46, and the VL comprises the sequence of SEQ ID NO: 47.

[0022] In some embodiments, the VH comprises the sequence of SEQ ID NO: 48, and the VL comprises the sequence of SEQ ID NO: 49.

[0023] In some embodiments, the VH comprises the sequence of SEQ ID NO: 65, and the VL comprises the sequence of SEQ ID NO: 49.

[0024] In some embodiments, the VH comprises the sequence of SEQ ID NO: 50, and the VL comprises the sequence of SEQ ID NO: 51.

[0025] In some embodiments, the VH comprises the sequence of SEQ ID NO: 63, and the VL comprises the sequence of SEQ ID NO: 51.

[0026] In some embodiments, the VH comprises the sequence of SEQ ID NO: 64, and the VL comprises the sequence of SEQ ID NO: 51.

[0027] In some embodiments, the VH comprises the sequence of SEQ ID NO: 52, and the VL comprises the sequence of SEQ ID NO: 53.

[0028] In some embodiments, the VH comprises the sequence of SEQ ID NO: 54, and the VL comprises the sequence of SEQ ID NO: 55.

[0029] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TIGIT, comprising: The heavy chain variable region (VH) comprises VH CDR1, VH CDR2, and VH CDR3 identical to those of the selected VH sequence; and the light chain variable region (VL) comprises VL CDR1, VL CDR2, and VL CDR3 identical to those of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO: 47; (2) The selected VH sequence is SEQ ID NO: 48 or 65, and the selected VL sequence is SEQ ID NO: 49; (3) The selected VH sequence is SEQ ID NO: 50, and the selected VL sequence is SEQ ID NO: 51; (4) The selected VH sequence is SEQ ID NO: 63, and the selected VL sequence is SEQ ID NO: 51; (5) The selected VH sequence is SEQ ID NO: 64, and the selected VL sequence is SEQ ID NO: 51; (6) The selected VH sequence is SEQ ID NO: 52, and the selected VL sequence is SEQ ID NO: 53; and (7) The selected VH sequence is SEQ ID NO: 54, and the selected VL sequence is SEQ ID NO: 55.

[0030] In some embodiments, the antibody or its antigen-binding fragment binds specifically to human or monkey TIGIT.

[0031] In some embodiments, the antibody or its antigen-binding fragment is a human or humanized antibody or its antigen-binding fragment.

[0032] In some implementations, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0033] In one respect, this disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.

[0034] In one aspect, this disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1-3, respectively, and wherein the VH binds TIGIT when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO: 47. (2) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL), wherein the VL comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 4-6, and wherein the VL binds TIGIT when paired with a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO: 46. (3) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 7-9, respectively, and wherein the VH binds TIGIT when paired with VL having an amino acid sequence as shown in SEQ ID NO: 49; (4) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 10-12, respectively, and wherein the VL binds TIGIT when paired with VH having amino acid sequences as shown in SEQ ID NO: 48 or 65; (5) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13-15, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (6) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16-18, and wherein the VL binds TIGIT when paired with VH having an amino acid sequence as shown in SEQ ID NO: 50; (7) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13, 61 and 15, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (8) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16-18, and wherein the VL binds TIGIT when paired with VH having an amino acid sequence as shown in SEQ ID NO: 63; (9) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13, 62 and 15, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (10) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:16-18, respectively, and wherein the VL binds TIGIT when paired with VH having an amino acid sequence as shown in SEQ ID NO:64; (11) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:19-21, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO:53; (12) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:22-24, respectively, and wherein the VL binds TIGIT when paired with VH having an amino acid sequence as shown in SEQ ID NO:52; (13) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:25-27, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO:55; (14) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:28-30, respectively, and wherein the VL binds TIGIT when paired with VH having an amino acid sequence as shown in SEQ ID NO:54; (15) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:31-33, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO:47; (16) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:34-36, respectively, and wherein the VH binds TIGIT when paired with VL having an amino acid sequence as shown in SEQ ID NO:49; (17) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:37-39, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO:51; (18) An immunoglobulin heavy chain or fragment thereof comprising VH, wherein the VH comprises CDR1, 2, and 3 having amino acid sequences as shown in SEQ ID NO:40-42, respectively, and wherein the VH binds TIGIT when paired with a VL having an amino acid sequence as shown in SEQ ID NO:53; and (19) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO:43-45, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO:55.

[0035] In some implementations, the VH specifically binds to human or monkey TIGIT when paired with VL.

[0036] In some embodiments, the immunoglobulin heavy chain or a fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or a fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.

[0037] In some implementations, the nucleic acid encodes a single-stranded variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0038] In some implementations, the nucleic acid is cDNA.

[0039] In one respect, this disclosure relates to a vector comprising one or more nucleic acids described herein.

[0040] In one aspect, this disclosure relates to a vector comprising two nucleic acids described herein, wherein in some embodiments, the vector encodes a VH region and a VL region that together bind TIGIT.

[0041] In one aspect, this disclosure relates to a pair of vectors, in some embodiments of which each vector contains one of the nucleic acids described herein, and in some embodiments of which the VH region and the VL region encoded by the pair of vectors are jointly bound to TIGIT.

[0042] In one respect, this disclosure relates to a cell, said cell including the vector described herein.

[0043] In some implementations, the cells are CHO cells.

[0044] In one respect, this disclosure relates to a cell comprising one or more nucleic acids described herein.

[0045] In one aspect, this disclosure relates to a method for generating an antibody or an antigen-binding fragment thereof, the method comprising:

[0046] (a) The cells are cultured under conditions sufficient to induce the cells described herein to produce the antibody or its antigen-binding fragment; and

[0047] (b) Collect antibodies or antigen-binding fragments thereof produced by the cells.

[0048] In one aspect, this disclosure relates to an antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to an antibody or its antigen-binding fragment described herein. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

[0049] In one aspect, this disclosure relates to a method of treating a subject suffering from cancer, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0050] In some implementations, the cancer is melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC)), colon cancer, hepatocellular carcinoma (HCC), stomach cancer, breast cancer, pancreatic cancer, glioblastoma, hematologic malignancy, kidney cancer, or ovarian cancer.

[0051] In some implementations, the cancer is melanoma, non-small cell lung cancer (NSCLC), colon cancer, hepatocellular carcinoma (HCC), gastric cancer, glioblastoma, hematologic malignancy, kidney cancer, or ovarian cancer.

[0052] In some embodiments, the method further includes administering to the subject a therapeutically effective amount of an anti-PD1 antibody, an anti-PDL1 antibody, an anti-PDL2 antibody, an anti-LAG-3 antibody, an anti-OX40 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, an anti-4-1BB antibody, and / or an anti-CD40 antibody.

[0053] In some implementations, the method further includes administering chemotherapy to the subject.

[0054] In one aspect, this disclosure relates to a method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0055] In one aspect, this disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0056] In one aspect, this disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

[0057] (a) The antibodies or antigen-binding fragments thereof described herein. (b) Antibody-drug conjugates described herein.

[0058] As used herein, the term “cancer” refers to cells capable of autonomous growth. Some examples of such cells include cells exhibiting an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growths, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of their histopathological type or degree of invasion. It also includes malignancies of various organ systems, such as the head and neck, respiratory, cardiovascular, renal, reproductive, hematopoietic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, gliomas, and small bowel cancers. “Naturally occurring” cancer includes any cancer that is not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously arising cancers, cancers caused by patient exposure to carcinogens, cancers caused by the insertion of oncogenes or the knockout of tumor suppressor genes, and cancers caused by infections, such as viral infections. The term "carcinoma" is generally accepted in the art and refers to a malignant disease of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to cancer originating from glandular tissue or in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted in the art and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can be caused by cells of the bone marrow, lymphatic system, or erythroid lineage or their precursors. Blood cancers are cancers originating from hematopoietic tissues (such as bone marrow) or immune system cells. Examples of blood cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0059] As used herein, the term "antibody" is used in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a protein complex comprising two heavy chains and two light chains. Other examples of antibodies are described herein.

[0060] As used herein, the terms "antigen-binding fragment" or "antibody fragment" refer to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment includes at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain, or VHH). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0061] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid derived from a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, human antibodies are collected from humans or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0062] As used herein, the term "humanized antibody" refers to a nonhuman antibody that contains a minimal sequence derived from a nonhuman (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. In some non-limiting examples, a humanized antibody is a human antibody (receptor antibody) in which hypervariable (e.g., CDR) region residues of the receptor antibody are replaced with hypervariable (e.g., CDR) region residues of a nonhuman antibody (e.g., donor antibody) (e.g., mouse, rat, or rabbit antibody) having the desired specificity, affinity, and capability. In some embodiments, Fv framework residues of a human immunoglobulin are replaced with corresponding nonhuman (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody may contain residues not present in the receptor antibody or donor antibody. These modifications may be made to further improve antibody performance. In some embodiments, a humanized antibody contains at least one and typically substantially all of two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to those of nonhuman (e.g., mouse) immunoglobulins, and all or substantially all of the framework regions are those of human immunoglobulins. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region (Fc) of human immunoglobulins. Humanized antibodies can be generated using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0063] As used herein, the term "multispecific antibody" refers to an antibody that binds to two or more different epitopes. Epitopes can be on the same antigen or different antigens. In some embodiments, multispecific antibodies are bispecific antibodies.

[0064] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. Epitopes can be on the same antigen or on different antigens.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials used in this invention are described herein; however, other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification (including definitions) shall prevail.

[0066] Other features and advantages of the invention will become apparent from the following detailed description, accompanying drawings, and claims. Attached Figure Description

[0067] Figure 1 The Kabat CDR sequence of the anti-TIGIT antibody is listed.

[0068] Figure 2 The Chothia CDR sequence for the anti-TIGIT antibody is listed.

[0069] Figure 3 The amino acid sequences discussed in this disclosure are listed.

[0070] Figure 4A This demonstrates the effect of blocking the binding of TIGIT to CD155 in the presence of anti-TIGIT antibodies (5F1 or 9H11). PC was used as a control.

[0071] Figure 4B This demonstrates the effect of blocking the binding of TIGIT to CD155 in the presence of anti-TIGIT antibodies (36F3 or 36H7). PC was used as a control.

[0072] Figure 4C This demonstrates the effect of blocking the binding of TIGIT to CD155 in the presence of anti-TIGIT antibody 15C5. PC was used as a control.

[0073] Figure 5 The concentration-time curves of anti-TIGIT antibody in different groups of C57BL / 6 mice are shown.

[0074] Figure 6 The ADCC activity assay results of the anti-TIGIT antibody are shown. PC was used as a control.

[0075] Figure 7The mean tumor volume of hTIGIT mice injected with MC38 cells and treated with PC (G2), 36F3 (G3), 36H7 (G4), or 9H11 (G5) is shown. PBS was used as a control (G1).

[0076] Figure 8 The mean tumor volume of hTIGIT mice injected with MC38 cells and treated with PC (G2), 9H11 (G3, G4), or 36F3 (G5, G6) is shown. PBS was used as a control (G1).

[0077] Figure 9 The mean tumor volume of hTIGIT mice in different groups injected with MC38 cells and treated with PC (G2) or 15C5 (G3) is shown. PBS was used as a control (G1). Detailed Implementation

[0078] This disclosure relates to anti-TIGIT antibodies or antigen-binding fragments thereof, and antibody-drug conjugates.

[0079] TIGIT

[0080] TIGIT (a T-cell immune receptor with Ig and ITIM domains, also known as WUCAM, Vstm3, or VSIG9) is a receptor of the Ig superfamily that plays a crucial role in limiting adaptive and innate immunity. TIGIT participates in a complex regulatory network involving multiple receptors (IRs) (e.g., CD96 / TACTILE, CD112R / PVRIG), a competitive co-stimulatory receptor (DNAM-1 / CD226), and multiple ligands (e.g., CD155 (PVR / NECL-5), CD112 (Nectin-2 / PVRL2)). Therefore, it shares some similarities with the CD28 / CTLA-4 / CD80 / CD86 pathway, in which inhibitory and co-stimulatory receptors compete for binding to the same ligands. (Similar to CTLA-4...) - / - In contrast, in mice, TIGIT - / - Mice do not develop autoimmunity. However, compared to wild-type mice, TIGIT... - / - Mice immunized with myelin oligodendrocyte glycoprotein developed more severe experimental autoimmune encephalitis. These observations support the role of TIGIT as a negative regulator of T cell function.

[0081] In humans, TIGIT is expressed by activated CD8+ and CD4+ T cells, natural killer (NK) cells, regulatory T cells (Tregs), and follicular helper T cells. In contrast to DNAM-1 / CD226, naïve T cells express only weakly TIGIT. In cancer, TIGIT is co-expressed with PD-1 on tumor antigen-specific CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) in mice and humans. It is also co-expressed with other immunoglobulins (IRs) on tumor-exhausted CD8+ T cell subsets, such as T cell immunoglobulin and mucin domain molecule-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3). Furthermore, TIGIT is highly expressed on regulatory T cells in peripheral blood mononuclear cells from healthy donors and cancer patients, and is further upregulated in the tumor microenvironment (TME).

[0082] TIGIT consists of an extracellular immunoglobulin (Ig) variable domain, a type I transmembrane domain, and a cytoplasmic tail conserved in mice and humans, bearing two inhibitory motifs: an immunoreceptor tyrosine inhibitory motif (ITIM) and an Ig-tail tyrosine (ITT)-like motif. The crystal structure of TIGIT bound to CD155 shows that two TIGIT / CD155 dimers assemble into a heterotetramer with a TIGIT / TIGIT cis homodimer at its core, with each TIGIT molecule binding to one CD155 molecule. This cis-trans receptor aggregation mediates cell adhesion and signal transduction.

[0083] The role of TIGIT in cancer is described in, for example, Chauvin, Joe-Marc, and Hassane M. Zarour, "TIGIT in cancer immunotherapy." Journal for immunotherapy of cancer 8.2 (2020); Harjunpää, H., and C. Guillerey, "TIGIT as an emerging immune checkpoint." Clinical & Experimental Immunology 200.2 (2020): 108-119; Rotte, Anand, Srikumar Sahasranaman, and Nageshwar Budha, "Targeting TIGIT for immunotherapy of cancer: Update on clinical development." Biomedicines 9.9 (2021): 1277, all of which are incorporated herein by reference in their entirety.

[0084] Anti-TIGIT antibody and antigen-binding fragment

[0085] This disclosure provides several antibodies that specifically bind to TIGIT and their antigen-binding fragments. The antibodies and antigen-binding fragments described herein are capable of binding to TIGIT. This disclosure provides, for example, anti-TIGIT antibodies 5F1, 9H11, 15C5, 36F3, and 36H7, and antibodies derived therefrom (e.g., 15C5-ND, 15C5-SG).

[0086] The CDR sequences of 5F1 and 5F1-derived antibodies (e.g., fully human antibodies) include the CDRs of the heavy chain variable domain (SEQ ID NO: 1-3) and light chain variable domains (SEQ ID NO: 4-6) as defined by Kabat. CDRs can also be defined by the Chothia system. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 31-33, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 4-6. The fully human heavy chain variable region and the fully human light chain variable region of 5F1 are shown in SEQ ID NO: 46 and SEQ ID NO: 47, respectively.

[0087] The CDR sequences of 9H11 and 9H11-derived antibodies (e.g., fully human antibodies) contain the CDRs of the heavy chain variable domain (SEQ ID NO: 7-9) and light chain variable domains (SEQ ID NO: 10-12) as defined by Kabat. Under the definition of Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 34-36, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 10-12. The fully human heavy chain variable region and the fully human light chain variable region of 9H11 are shown in SEQ ID NO: 65 and SEQ ID NO: 49, respectively.

[0088] The CDR sequences of 15C5 and 15C5-derived antibodies (e.g., fully human antibodies) include the CDRs of the heavy chain variable domain (SEQ ID NO: 13-15) and the light chain variable domain (SEQ ID NO: 16-18) as defined by Kabat. Under the definition of Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 37-39, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 16-18. The fully human heavy chain variable region and the fully human light chain variable region of 15C5 are shown in SEQ ID NO: 50 and SEQ ID NO: 51, respectively.

[0089] The CDR sequence of the 15C5-derived antibody 15C5-ND includes the CDRs of the heavy chain variable domain (SEQ ID NO: 13, 61, 15) and the light chain variable domain (SEQ ID NO: 16-18) as defined by Kabat. Under the definition of Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 37-39, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 16-18. The fully human heavy chain variable region and the fully human light chain variable region of 15C5-ND are shown in SEQ ID NO: 63 and SEQ ID NO: 51, respectively.

[0090] The CDR sequence of the 15C5-derived antibody 15C5-SG includes the CDRs of the heavy chain variable domain (SEQ ID NO: 13, 62, 15) and the light chain variable domain (SEQ ID NO: 16-18) as defined by Kabat. Under the definition of Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 37-39, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 16-18. The fully human heavy chain variable region and the fully human light chain variable region of 15C5-SG are shown in SEQ ID NO: 64 and SEQ ID NO: 51, respectively.

[0091] The CDR sequences of 36F3 and 36F3-derived antibodies (e.g., fully human antibodies) contain the CDRs of the heavy chain variable domain (SEQ ID NO: 19-21) and light chain variable domains (SEQ ID NO: 22-24) as defined by Kabat. Under the definition of Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 40-42, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 22-24. The fully human heavy chain variable region and the fully human light chain variable region of 36F3 are shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

[0092] The CDR sequences of 36H7 and 36H7-derived antibodies (e.g., fully human antibodies) contain the CDRs of the heavy chain variable domain (SEQ ID NO: 25-27) and light chain variable domains (SEQ ID NO: 28-30) as defined by Kabat. Under the definition of Chothia, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 43-45, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 28-30. The fully human heavy chain variable region and the fully human light chain variable region of 36H7 are shown in SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

[0093] Furthermore, in some embodiments, the antibody or its antigen-binding fragment described herein may also include one, two, or three heavy chain variable regions (CDRs) selected from SEQ ID NO: 1, 2, 3; SEQ ID NO: 7, 8, 9; SEQ ID NO: 13, 14, 15; SEQ ID NO: 19, 20, 21; SEQ ID NO: 25, 26, 27; SEQ ID NO: 31, 32, 33; SEQ ID NO: 34, 35, 36; SEQ ID NO: 37, 38, 39; SEQ ID NO: 40, 41, 42 and SEQ ID NO: 43, 44, 45; and / or one, two, or three light chain variable regions (CDRs) selected from SEQ ID NO: 4, 5, 6; SEQ ID NO: 10, 11, 12; SEQ ID NO: 16, 17, 18; SEQ ID NO: 22, 23, 24 and SEQ ID NO: 28, 29, 30.

[0094] In some embodiments, the antibody may have: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the selected VH CDR1 amino acid sequence; CDR2 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the selected VH CDR2 amino acid sequence; and CDR3 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein CDR1 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the selected VL CDR1 amino acid sequence; and CDR2 ...L CDR1 amino acid sequence; and CDR2 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the selected VH CDR1 amino acid sequence; and CDR3 contains or is composed of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with the selected VH CDR1 amino acid sequence; and CDR2 contains or is The CDR2 amino acid sequence has or is composed of an amino acid sequence with at least 80%, 85%, 90%, or 95% identity with the selected VL CDR3 amino acid sequence, and the CDR3 region contains or is composed of an amino acid sequence with at least 80%, 85%, 90%, or 95% identity with the selected VL CDR3 amino acid sequence. The selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL CDR 1, 2, and 3 amino acid sequences are as follows: Figure 1 (Kabat CDR) and Figure 2 As shown in (Chothia CDR).

[0095] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 1 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 3 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0096] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 7 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 9 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0097] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 13 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 15 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0098] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 13 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 61 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 15 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0099] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 13 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 15 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0100] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0101] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 25 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 27 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0102] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 31 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 33 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0103] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 34 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 36 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0104] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 37 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 39 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0105] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 40 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 42 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0106] In some embodiments, the antibody or antigen-binding fragment described herein may include a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 43 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 45 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0107] In some embodiments, the antibody or antigen-binding fragment described herein may include a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 4 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 6 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0108] In some embodiments, the antibody or antigen-binding fragment described herein may include a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0109] In some embodiments, the antibody or antigen-binding fragment described herein may include a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 16 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 18 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0110] In some embodiments, the antibody or antigen-binding fragment described herein may include a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 22 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 24 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0111] In some embodiments, the antibody or antigen-binding fragment described herein may include a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 28 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 having 0, 1, or 2 amino acid insertions, deletions, or substitutions; and SEQ ID NO: 30 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0112] Insertions, deletions, and substitutions can occur within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on the Kabat definition. In some embodiments, the CDR is determined based on the Chothia definition. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia definitions. In some embodiments, the CDR is determined based on the IMGT definition.

[0113] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to TIGIT. The antibody or antigen-binding fragment comprises: a heavy chain variable region (VH) containing or consisting of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VH sequence; and a light chain variable region (VL) containing or consisting of an amino acid sequence having at least 80%, 85%, 90%, or 95% identity with a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 46, 48, 50, 52, 54, 63, 64, or 65, and the selected VL sequence is SEQ ID NO: 47, 49, 51, 53, or 55.

[0114] In some embodiments, the antibody or its antigen-binding fragment may have the same three VH CDRs as any VH sequence described herein. In some embodiments, the antibody or its antigen-binding fragment may have the same three VL CDRs as any VL sequence described herein.

[0115] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides, said polypeptides comprising immunoglobulin heavy chains or immunoglobulin light chains. The immunoglobulin heavy chains or immunoglobulin light chains comprise, for example... Figure 1 or Figure 2 The CDR shown, or having as Figure 3 The sequence is shown. When the polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), the paired polypeptide binds to TIGIT (e.g., human TIGIT).

[0116] Anti-TIGIT antibodies and antigen-binding fragments can also be variants of antibodies or antibody fragments (including derivatives and conjugates), and multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies described herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (multimeric antibodies, such as bispecific antibodies), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly generated antibodies (i.e., intrabody antibodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG (e.g., IgG1) antibody or its antigen-binding fragment.

[0117] Antibody fragments are suitable for use in the methods presented herein, provided they retain the desired affinity and specificity of the full-length antibody. Therefore, fragments of antibodies that bind TIGIT will retain the ability to bind TIGIT. Fv fragments are antibody fragments containing complete antigen recognition and binding sites. This region consists of a dimer of a tightly associated heavy-chain variable domain and a light-chain variable domain, the association being covalent in nature, as in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six CDRs, or a subset thereof, collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind antigens, but typically with lower affinity than the entire binding site.

[0118] This invention also provides antibodies or antigen-binding fragments thereof capable of cross-competing with any antibody or antigen-binding fragment described herein. Cross-competition analysis is known in the art, for example, as described in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein", Journal of Virology, 70.3 (1996): 1863-1872, the entire text of which is incorporated herein by reference. In one aspect, this invention also provides antibodies or antigen-binding fragments thereof capable of binding to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope sorting analysis is known in the art, for example, as described in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning", MAbs, Vol. 5, No. 2, Taylor & Francis, 2013, the entire text of which is incorporated herein by reference.

[0119] Antibody and antigen binding fragment

[0120] This disclosure provides antibodies derived from the anti-TIGIT antibodies described herein and their antigen-binding fragments. Typically, antibodies (also called immunoglobulins) can be composed of two types of polypeptide chains: light chains and heavy chains. The non-limiting antibody of this disclosure can be a complete tetraimmunoglobulin chain antibody containing two heavy chains and two light chains. The heavy chain of the antibody can be any isotype, including IgM, IgG, IgE, IgA, or IgD; or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. The antibody may contain two identical copies of the light chain and / or two identical copies of the heavy chain. Each contains a variable domain (or variable region, V...). H The heavy chain, consisting of multiple constant domains (or constant regions), is linked together by disulfide bonds within its constant domains to form the "stem" of the antibody. Each heavy chain contains a variable domain (or variable region, V...). L Each of the light chains, consisting of a light chain and a constant structural domain (or constant region), is bonded to a heavy chain via disulfide bonds. The variable region of each light chain pairs with the variable region of the heavy chain it is bonded to. The variable regions of both the light and heavy chains contain three highly variable regions located between the more conservative framework regions (FRs).

[0121] These are called complementarity-determining regions (CDRs) and hypervariable regions, forming a loop containing the main antigen-binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form a loop structure that is connected and, in some cases, forms part of a β-sheet structure. The CDRs in each chain are kept close together by the framework regions and, together with CDRs from other chains, contribute to the formation of the antigen-binding region.

[0122] Identifying the CDR region of an antibody by analyzing its amino acid sequence is a well-known method, and many definitions of CDR are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in the following: for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecularimmunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp.Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., BiophysChem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan .1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each is incorporated into this paper in its entirety through citation.

[0123] CDR is important for recognizing antigenic epitopes. As used in this article, an "epitaph" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not necessarily have to be located in a continuous linear sequence of the primary antigen structure, as epitopes can depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0124] In some implementations, antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing only in their constant regions, particularly their hinges and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described, for example, in the following: Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecularimmunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0125] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camels). The antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin-binding domain fused to another polypeptide. An antigen-binding domain or antigen-binding fragment is an antibody portion that retains the specific binding activity of the intact antibody; that is, any portion of the antibody capable of specifically binding to an epitope on the target molecule of the intact antibody. These include, for example, Fab, Fab', F(ab')2, and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, biantibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies, and any polypeptide comprising an antibody-binding domain or a binding domain homologous to the antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy chain and / or light chain CDR of an intact antibody, the heavy chain and / or light chain variable region of an intact antibody, the full-length heavy chain or light chain of an intact antibody, or a single CDR from the heavy chain or light chain of an intact antibody.

[0126] Antibody fragments suitable for the methods described herein are also provided. The Fab fragments comprise variable and constant domains of the light chain and variable and first constant domains (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near their carboxyl ends by a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known in the art.

[0127] A biantibody is a small antibody fragment with two antigen-binding sites, contained within a VL-linked VH (VH and VL) on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains on the other chain, creating two antigen-binding sites.

[0128] Linear antibodies consist of a pair of tandem Fd regions (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0129] The antibodies and antibody fragments disclosed herein can be modified in the Fc region to provide desired effector function or serum half-life.

[0130] Antibody polymerization can be achieved through the natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates comprising antibody homodimers and other higher-order antibody polymers.

[0131] Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art. For example, heterobifunctional crosslinking agents can be used to form antibody polymers, including but not limited to SMCC (succinimide-4-(maleimide-methyl)cyclohexane-1-carboxylic acid ester) and SATA (N-succinimide-S-acetylthioacetate ester). Ghetie et al. ( Proc. Natl. Acad. Sci. USA Exemplary schemes for forming antibody homodimers are described in 94: 7509-7514, 1997. Antibody homodimers can be converted into Fab'2 homodimers by digestion with pepsin. Another method for forming antibody homodimers is by using Zhao et al. ( J. Immunol. The self-affinity T15 peptide described in 25:396-404, 2002.

[0132] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be prepared by modifying the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size targeting the large side chains are generated at the interface of the second antibody molecule by replacing the larger amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products (e.g., homodimers). This method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0133] Antibodies or their antigen-binding fragments can also take various forms. Many different formats of antigen-binding constructs are known in the art and described, for example, in Suurs et al., "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges," Pharmacology & Therapeutics (2019), which is incorporated herein by reference in its entirety.

[0134] Bispecific antibodies include cross-linked antibodies or "isoconjugated" antibodies. For example, one antibody in an isoconjugation may be conjugated to avidin, and another to biotin. Isoconjugated antibodies can also be prepared using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0135] Any antibody or antigen-binding fragment described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of an antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation to a stabilizing molecule may prolong the half-life of the antibody or antigen-binding fragment in vitro (e.g., in tissue cultures or when stored as a pharmaceutical composition) or in vivo (e.g., in humans) or enhance its biological activity.

[0136] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of the single-stranded variable fragment (scFv) described herein, fused with both the CD3-ζ transmembrane and intracellular domains. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from multiple co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to enhance potency. Therefore, in one aspect, this disclosure also provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0137] In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be conjugated to a therapeutic agent. Antibody-pharmaceutical conjugates comprising antibodies or antigen-binding fragments thereof may covalently or non-covalently bind a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., monomethylaurestatin E, monomethylaurestatin F, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxine, maytansine alkaloids (such as DM-1 and DM-4), diketones, mitoxantrone, styracin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues). In some embodiments, the therapeutic agent is MMAE or MMAF. In some implementations, the therapeutic agent is coupled via a linker (e.g., a VC linker). For more information on linkers used in ADCs, see Su, Z et al., "Antibody–drug conjugates: Recent advances in linker chemistry." Acta Pharmaceutica Sinica B (2021), the full text of which is incorporated herein by reference.

[0138] In some embodiments, the sequences of the antibodies described herein or their antigen-binding fragments (e.g., CDR or VH / VL sequences) can be used to generate bispecific antibodies targeting TIGIT and additional antigens (e.g., OX40, CD40, 4-1BB, CTLA4, PD-1, or PD-L1).

[0139] Antibody characteristics

[0140] This disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to TIGIT. The antibodies and antigen-binding fragments described herein can bind to TIGIT. These antibodies can be agonists or antagonists. In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment described herein can bind to TIGIT and block the binding between TIGIT and CD155 and / or between TIGIT and CD112. By binding to TIGIT, the anti-TIGIT antibody can downregulate or upregulate TIGIT-related signaling pathways. In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment described herein can bind to TIGIT but does not block the binding between TIGIT and CD155 and / or between TIGIT and CD112. In some embodiments, the antibody or its antigen-binding fragment described herein is a TIGIT agonist. In some embodiments, the antibody or its antigen-binding fragment is a TIGIT antagonist.

[0141] The affinity of an antibody for an antigen can be measured using common techniques, including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and surface plasmon resonance (SPR). Affinity can be derived as the quotient of the kinetic rate constant (KD = koff / kon). In some embodiments, the antibody or its antigen-binding fragment can specifically bind TIGIT (e.g., human TIGIT, monkey TIGIT, mouse TIGIT, and / or chimeric TIGIT) at the following dissociation rate (koff): less than 0.1 s. -1 Less than 0.01 s -1 Less than 0.001 s -1 Less than 0.0001 s -1 or less than 0.00001 s -1 In some implementations, the dissociation rate (koff) is greater than 0.01 s⁻¹. -1 Greater than 0.001 s -1 Greater than 0.0001 s -1 Greater than 0.00001 s -1 or greater than 0.000001 s -1 .

[0142] In some implementations, the kinetic association rate (kon) is greater than 1 x 10⁻⁶. 2 / Ms, greater than 1 x 10 3 / Ms, greater than 1 x 10 4 / Ms, greater than 1 x 10 5 / Ms or greater than 1 x 10 6 / Ms. In some implementations, the kinetic association rate (kon) is less than 1 x 10 5 / Ms, less than 1 x 10 6 / Ms or less than 1 x 10 7 / Ms.

[0143] In some embodiments, the antibody or its antigen-binding fragment may bind to TIGIT (e.g., human TIGIT, monkey TIGIT, mouse TIGIT, and / or chimeric TIGIT) with the following KD values: less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M or less than 1 x 10 -10M. In some embodiments, KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10⁻⁶. -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M or greater than 1 x 10 -10 M.

[0144] In some embodiments, the tumor growth inhibition rate or percentage (TGI%) of the antibody or its antigen-binding fragment is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the tumor growth inhibition percentage of the antibody is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150%. TGI (%) may be determined, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days after the start of treatment. As used herein, the tumor growth inhibition rate or percentage (TGI%) is calculated using the following formula: TGI (%) = [1-T i -T0V i -V0]×100 Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0145] In some embodiments, the antibody or its antigen-binding fragment can increase the number of tumor-infiltrating lymphocytes. In some embodiments, the antibody or its antigen-binding fragment can increase the number of tumor-infiltrating lymphocytes by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%.

[0146] In some embodiments, the antibody or its antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are both ADCC and phagocytosis. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0147] In some embodiments, the antibody or its antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the antibody or its antigen-binding fragment described herein has an effector-free Fc region. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbers) or LALA-PG mutations (L234A, L235A, P329G mutations according to EU numbers).

[0148] The Fc region can undergo several other modifications. For example, cysteine ​​residues can be introduced into the Fc region, allowing interchain disulfide bonds to form there. The resulting homodimeric fusion protein may have an increased half-life in vitro and / or in vivo.

[0149] In some embodiments, an Fc region with a sugar structure is provided that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an Fc region composition can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, as described, for example, in WO 2008 / 077546, by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, heterozygous, and high-mannose structures) attached to Asn297, by MALDI-TOF mass spectrometry. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu number of the Fc region residue; or position 314 in the Kabat number); however, due to minor sequence variations in the Fc region sequence, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylation variants can possess improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further modified to replace the asparagine at position 297 with alanine (N297A).

[0150] Methods for preparing anti-TIGIT antibodies

[0151] Isolated fragments of human proteins can be used as immunogens to generate antibodies using standard techniques for the preparation of polyclonal and monoclonal antibodies. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to a reagent that is immunogenic in the species to be immunized. The antigenic peptide or protein may be injected into the animal more than once (e.g., twice, three times, four times, or five times).

[0152] Full-length polypeptides or proteins can be used, or alternatively, their antigenic peptide fragments can be used as immunogens. The antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the protein's amino acid sequence and contains the protein's epitope, such that antibodies generated against the peptide form specific immune complexes with the protein.

[0153] Immunogens are commonly used to prepare antibodies by immunizing suitable subjects (e.g., humans or transgenic animals expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed or chemically synthesized peptides. The preparation may further contain adjuvants, such as Freund's complete or incomplete adjuvants, or similar immunostimulants.

[0154] Polyclonal antibodies can be prepared by immunizing suitable subjects with a polypeptide or its antigenic peptide (e.g., a fraction of a protein) as an immunogen, as described above. Antibody titers in immunized subjects can be monitored over time using standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptides or peptides. If desired, antibody molecules can be isolated from mammals (e.g., from blood) and further purified using known techniques such as protein A or protein G chromatography to obtain the IgG fraction. At an appropriate time following immunization, e.g., when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies using standard techniques, such as those employed by Kohler et al. Nature Hybridoma technology, originally described in 256:495-497, 1975, and human B-cell hybridoma technology (Kozbor et al., Immunol. Today4:72, 1983), EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma technology. Techniques for generating hybridomas are well-known (generally see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected, for example, by screening for antibodies binding to target peptides or epitopes in the supernatant of hybridoma cultures using standard ELISA assays.

[0155] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide alterations into the DNA encoding the human antibodies, humanized antibodies, or chimeric antibodies or their antigen-binding fragments, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence constituting the antigen-binding site of the antibody or antigen-binding domain. Within a population of such variants, some antibody or antigen-binding fragments will have enhanced affinity for the target protein. Any combination of deletions, insertions, and / or combinations can be made to obtain antibodies or their antigen-binding fragments with enhanced binding affinity to the target. Introducing amino acid changes into the antibody or antigen-binding fragment can also alter the antibody or antigen-binding fragment or introduce new post-translational modifications therein, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence to allow different sugars to attach via enzymes present in the cell), or introducing new glycosylation sites.

[0156] The antibodies disclosed herein may be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include those derived from: humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camels (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including genetically modified rodents that have been genetically engineered to produce human antibodies.

[0157] Phage display (panning) can be used to optimize antibody sequences with desired binding affinity. In this technique, a gene encoding a single-stranded Fv (including VH or VL) is inserted into a phage coat protein gene, causing the phage to "display" scFv on its outer side while containing the gene for that protein inside, thus establishing a link between genotype and phenotype. These displaying phages can then be screened against a target antigen to detect the interaction between the displayed antigen-binding site and the target antigen. Therefore, large protein libraries can be screened and amplified in a process called in vitro selection, and antibody sequences with desired binding affinity can be obtained.

[0158] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequences as those derived therefrom). Human antibodies may contain amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo), such as in CDRs.

[0159] Humanized antibodies typically possess a human frame (FR) with transplanted non-human CDRs. Therefore, humanized antibodies have one or more amino acid sequences introduced from a non-human source. Thus, "humanized" antibodies are chimeric antibodies, where essentially less than the complete human V domain has been replaced by corresponding sequences from a non-human species. In practice, humanized antibodies are often mouse antibodies, where some CDR residues and some FR residues are replaced by residues from similar sites in human antibodies.

[0160] Further important is the humanization of antibodies while retaining high specificity and affinity for antigens, as well as other advantageous biological properties. To achieve this, humanized antibodies can be prepared through a process of analyzing parental sequences and various conceptual humanization products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are universally available and well-known to those skilled in the art. Computer programs illustrating and demonstrating the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examination of these demonstrations allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequences, i.e., analysis of residues affecting the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from receptor and input sequences to obtain desired antibody characteristics, such as enhanced affinity for the target antigen.

[0161] In some embodiments, antibodies are generated using mice with humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenMab™ mice). A heavy chain immunoglobulin locus is a region of a chromosome containing an antibody heavy chain gene. This locus may include, for example, the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (connection) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a region of a chromosome containing an antibody light chain (κ chain) gene. The κ chain immunoglobulin locus may include, for example, the human IGKV (variable) gene, the human IGKJ (connection) gene, and the mouse light chain constant domain gene. A detailed description of the RenMab™ mouse can be found in PCT / CN2020 / 075698 or US20200390073A1, the full text of which is incorporated herein by reference.

[0162] Identity or homology relative to the original sequence is typically the percentage of amino acid residues in a candidate sequence that are identical to those present in a human, humanized, or chimeric antibody or fragment, provided that the sequence is aligned and gaps are introduced (if necessary) to achieve the maximum percentage of sequence identity and no conserved substitutions are considered part of the sequence identity.

[0163] In some embodiments, antibodies or their antigen-binding fragments can be covalently modified. These covalent modifications can be performed through chemical or enzymatic synthesis, or through enzymatic or chemical cleavage. Other types of covalent modifications to antibodies or antibody fragments can be introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizer capable of reacting with selected side chains or N- or C-terminal residues.

[0164] In some embodiments, antibody variants are provided that have a carbohydrate structure lacking (directly or indirectly) fucose linked to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures linked to Asn297 (e.g., complex, heterozygous, and high-mannose structures), as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to the asparagine residue located approximately at position 297 in the Fc region (Eu number of the Fc region residue; or position 314 in the Kabat number); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants may possess improved ADCC function. In some implementations, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the asparagine at position 297 (N297A) with alanine.

[0165] In some implementations, to improve production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further engineered by replacing the serine at position 228 (EU number) of IgG4 with proline (S228P). For a detailed description of the S228 mutation, see, for example, Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9 (2015): 5462-5469, the full text of which is incorporated herein by reference.

[0166] Recombinant vector

[0167] This disclosure also provides recombinant vectors (e.g., expression vectors) comprising isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vector is introduced (i.e., vectors containing polynucleotides such that the host cells contain polynucleotides and / or contain polynucleotides), and the generation of recombinant antibody polypeptides or fragments thereof via recombinant technology.

[0168] As used herein, a “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when introduced into the host cell. An “expression vector” is capable of delivering one or more target polynucleotides and expressing one or more target polynucleotides as encoded polypeptides in a host cell in which the expression vector has been introduced. Thus, in an expression vector, the target polynucleotide is positioned for expression in the vector by operatively linking to regulatory elements, such as promoters, enhancers, and / or poly-A tails, in or near the integration site of the target polynucleotide, such that the target polynucleotide will be translated in the host cell in which the expression vector has been introduced.

[0169] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection and infection, and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic coagulants.

[0170] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia virus or other poxviruses, retroviruses, or adenoviruses). This may involve using a non-pathogenic (deficient) replicable virus, or a replication-deficient virus. In the latter case, viral replication typically occurs only in complementary viral packaging cells. Suitable systems are disclosed in, for example, the following: Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; US ​​Pat. No. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; US Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be enhanced by coating DNA onto biodegradable beads, which are then efficiently transported into cells.

[0171] For expression, a DNA insert containing a polynucleotide encoding an antibody or polypeptide disclosed herein can be efficiently linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, or the Escherichia coli (E. coli) promoter. E. coliPromoters such as lac, trp, and tac, early and late SV40 promoters, and promoters of retroviral LTRs are available. Other suitable promoters are known to those skilled in the art. The expression construct may further include sites for transcription initiation and termination, as well as ribosome-binding sites in the transcriptional region for translation. The coding portion of the mature transcript expressed by the construct may include a translation origin at the initiation site and a stop codon (UAA, UGA, or UAG) appropriately located at the end of the polypeptide to be translated.

[0172] As shown, the expression vector may contain at least one selection marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Some representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli*, *Streptomyces*, etc. Streptomyces ) and Salmonella typhimurium ( Salmonella typhimurium The host cells described herein include: fungal cells, such as yeast cells; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0173] Non-restrictive vectors for bacteria include pQE70, pQE60, and pQE-9, available from Qiagene; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5, available from Pharmacia. Non-restrictive eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0174] Suitable non-restrictive bacterial promoters include the *E. coli* lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, promoters of retroviral LTRs such as the Rous sarcoma virus (RSV) promoter, and metallothionein promoters such as the mouse metallothionein-I promoter.

[0175] In brewer's yeast ( Saccharomyces cerevisiae In this study, many vectors containing constitutive or inducible promoters can be used, such as α-factor, alcohol oxidase, and PGH. For reviews, see Ausubel et al. (1989), Current Protocols in Molecule Biology, John Wiley & Sons, New York, NY, and Grant et al. Methods Enzymol., 153: (1997).

[0176] Constructs can be introduced into host cells via calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0177] The transcription of DNA encoding the disclosed antibodies in higher eukaryotes can be enhanced by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, that enhance promoter transcriptional activity in a given host cell type. Some examples of enhancers include the SV40 enhancer located 100 to 270 base pairs post-OMS, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located post-OMS, and the adenovirus enhancer.

[0178] To facilitate the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment, appropriate secretion signals can be incorporated into the expressed peptide. These signals can be endogenous to the peptide or can be heterologous.

[0179] Peptides (e.g., antibodies) can be expressed in modified forms (e.g., fusion proteins, such as GST-fusions, or those with histidine tags) and may contain not only secretion signals but also additional heterologous functional regions. For example, additional amino acid regions (particularly charged amino acids) can be added to the N-terminus of the peptide to improve stability and persistence in host cells, during purification, or during subsequent processing and storage. Additionally, peptide moieties can be added to the peptide to facilitate purification. These regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to induce secretion or excretion, to improve stability, and to facilitate purification are well-known and conventional techniques in the art.

[0180] This disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with any nucleotide sequence as described herein, and amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with any amino acid sequence as described herein.

[0181] This disclosure also provides nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any nucleotide sequence as described herein, and amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any amino acid sequence as described herein.

[0182] In some embodiments, this disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, or 3000 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, or 800 amino acid residues.

[0183] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0184] In some implementations, the nucleic acid sequence (i) contains a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0185] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence (the term "identity" for amino acids or nucleic acids used herein is equivalent to "homology" for amino acids or nucleic acids). The percentage identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. For example, the comparison of sequences and the determination of the percentage identity between two sequences can be accomplished using a Blossum 62 scoring matrix, where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0186] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. Methods for determining the percentage of sequence homology are known in the art. In some embodiments, conserved amino acid residues (e.g., leucine and isoleucine) having similar physicochemical properties (homology percentage) can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in this art. These families include, for example, amino acids having basic branches (e.g., lysine, arginine, histidine), acidic branches (e.g., aspartic acid, glutamic acid), uncharged polar branches (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar branches (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched branches (e.g., threonine, valine, isoleucine), and aromatic branches (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percentage of homology is higher than the percentage of identity.

[0187] This disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding the heavy chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding the light chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding the scFv polypeptide described herein.

[0188] In some embodiments, the vector may have both of the nucleic acids described herein, wherein the vector encodes both the VL and VH regions of TIGIT that bind together. In some embodiments, a pair of vectors is provided, wherein each vector contains either of the nucleic acids described herein, wherein the pair of vectors together encodes both the VL and VH regions of TIGIT that bind together.

[0189] The vector can also be constructed to express specific antibodies or peptides. In some embodiments, the vector can be constructed to co-express the anti-TIGIT antibody light chain (TIGIT-K) and heavy chain (TIGIT-H). In some embodiments, the vector may contain, from the 5' to the 3' end, sequences of a cytomegalovirus promoter (CMV), TIGIT-K, polyadenylation (PolyA), CMV, TIGIT-H, PolyA, simian vacuolar virus 40 terminator (SV40), and glutamine synthetase marker (GS). In some embodiments, a first vector expressing the antibody heavy chain (e.g., any heavy chain described herein) and a second vector expressing the antibody light chain (e.g., any light chain described herein) are co-transfected into cells (e.g., CHO cells) to produce the monoclonal antibody or its antigen-binding fragment described herein.

[0190] Treatment

[0191] The methods described herein include methods for treating cancer-related conditions. Generally, the methods involve administering a therapeutically effective amount of the engineered antibody or antigen-binding fragment thereof described herein to a subject who needs or has been identified as needing such treatment.

[0192] As used herein, “treatment” means improvement of at least one symptom of a cancer-related condition. Cancer often leads to death; therefore, treatment can result in an increase in life expectancy (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). Administration of a therapeutically effective amount of the agents described herein for the treatment of a cancer-related condition will result in a reduction in the number of cancer cells and / or symptom relief.

[0193] As used herein, the term “cancer” refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. This term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of their histopathological type or stage of invasion. As used herein, the term “tumor” refers to cancer cells, such as a population of cancer cells. Cancers that can be treated or diagnosed using the methods described herein include: malignant tumors of various organ systems, such as those affecting the lungs, breast, thyroid, lymph nodes, gastrointestinal tract, and genitourinary tract; and adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell lung cancer, small bowel cancer, and esophageal cancer. In some embodiments, the reagents described herein are designed to treat or diagnose cancer in a subject. The term “cancer” is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some embodiments, the cancer is kidney cancer or melanoma. Exemplary cancers include those that originate from cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. This term also includes carcinosarcoma, such as malignant tumors comprising both cancerous and sarcomatous tissue. “Adenocarcinoma” refers to cancer originating from glandular tissue or where tumor cells form identifiable glandular structures. The term “sarcoma” is recognized in the art and refers to a mesenchymal-derived malignant tumor. In some embodiments, the cancer is lung cancer, skin cancer, head and neck cancer, bladder cancer, prostate cancer, breast cancer, endometrial cancer, cervical cancer, pancreatic cancer, glioma, lymphoma, or leukemia. In some embodiments, the cancer is melanoma, non-small cell lung cancer (NSCLC), colon cancer, hepatocellular carcinoma (HCC), gastric cancer, glioblastoma, hematologic malignancies, kidney cancer, or ovarian cancer.

[0194] In some implementations, the cancer is chemotherapy-resistant cancer.

[0195] In one aspect, this disclosure also provides methods for treating cancer in a subject, methods for reducing the rate at which the volume of a tumor increases over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of additional metastasis in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0196] In one aspect, this disclosure is characterized by a method comprising administering a therapeutically effective amount of the disclosed antibody, its antigen-binding fragment, or antibody-drug conjugate to a subject in need, such as a subject who has or has been identified or diagnosed with cancer, for example, a solid tumor, lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, or lung cancer), gastric cancer (e.g., stomach cancer), skin cancer (e.g., skin cancer), colorectal cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, thyroid cancer, pancreatic cancer, CNS cancer, liver cancer, nasopharyngeal cancer, or brain cancer.

[0197] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe human or non-human animals for which treatment is provided according to the method of the invention. This disclosure is contemplated for both veterinary and non-veterinary applications. Human patients can be adults or minors (e.g., persons under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic, farm, and zoo animals.

[0198] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients with cancer can be identified by a variety of methods known in the art.

[0199] As used herein, “effective amount” means an amount or dose sufficient to produce a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease such as cancer. Effective amounts will vary depending on, for example, the age and weight of the individual to whom the antibody, antigen-binding fragment, antibody-drug conjugate, polynucleotide encoding the antibody, carrier containing the polynucleotide, and / or combinations thereof are administered, the severity of symptoms, and the route of administration, and therefore may be determined on an individual basis.

[0200] An effective amount may be administered in one or more applications. For example, an effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient in vitro to improve, stop, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any cancer cells or cell lines described herein, e.g., cancer cell lines). As understood in the art, the effective amount of an antibody, antigen-binding fragment, or antibody-drug conjugate can vary, particularly depending on the patient's medical history and other factors such as the type (and / or dosage) of the antibody used.

[0201] The effective amount and schedule for administering the antibodies, polynucleotides encoding antibodies, antibody-drug conjugates, and / or compositions disclosed herein are determined empirically, and such determination is within the scope of the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal to which the antibodies, polynucleotides encoding antibodies, antibody-drug conjugates, and / or compositions disclosed herein will be received, the route of administration, the specific type of antibody, polynucleotide encoding antibody, antigen-binding fragment, antibody-drug conjugate, and / or composition disclosed herein used, and any other drugs administered to the mammal. Guidelines for selecting appropriate doses of antibodies or antigen-binding fragments can be found in literature on the therapeutic uses of antibodies and antigen-binding fragments, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0202] The typical daily dose of an antibody, its antigen-binding fragment, or antibody-drug conjugate is from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may be less than 100 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some implementations, the dosage is approximately 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0203] In any of the methods described herein, at least one antibody, its antigen-binding fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any antibody, antigen-binding fragment, or antibody-drug conjugate described herein), and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody, its antigen-binding fragment, or antibody-drug conjugate, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered in the form of pills, tablets, or capsules. In some embodiments, the at least one additional therapeutic agent is a sustained-release oral formulation.

[0204] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein). In some embodiments, administration of one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) to a subject results in an overlap in the biological activity periods of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any antibody or antigen-binding fragment described herein).

[0205] In some implementations, at least one antibody, antigen-binding antibody fragment, antibody-drug conjugate, or pharmaceutical composition (e.g., any one of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) may be administered to a subject over an extended period of time (e.g., at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). Skilled medical professionals may use any of the methods described herein to determine the length of the treatment period for diagnosis or to track treatment effectiveness (e.g., to observe at least one symptom of cancer). As described herein, skilled medical professionals may also vary the type and quantity (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment, antibody-drug conjugate (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of treatment effectiveness (e.g., using any of the methods described herein and known in the art).

[0206] In some embodiments, one or more additional therapeutic agents may be administered to the subject. These additional therapeutic agents may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, PD-1 inhibitors, CD155 inhibitors, CD112 inhibitors, 4-1BB inhibitors, CD40 inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0207] In some implementations, additional therapeutic agents may include one or more inhibitors selected from the group consisting of: TIGIT inhibitors, CD155 inhibitors, CD112 inhibitors, 4-1BB inhibitors, CD40 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0208] In some implementations, additional therapeutic agents may include one or more agents selected from the group consisting of: Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, and sorafenib. nib), Votrient, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0209] In some implementations, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0210] In some implementations, subjects are administered carboplatin, albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0211] In some implementations, additional therapeutic agents are anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-LAG-3 antibodies, anti-BTLA antibodies, anti-CTLA4 antibodies, anti-CD40 antibodies, anti-OX40 antibodies, anti-4-1BB antibodies, or anti-GITR antibodies.

[0212] Pharmaceutical Compositions and Routes of Administration

[0213] This document also provides pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) of the antibody or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the above-described antibody or antigen-binding fragments may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0214] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), fixative oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetates, citrates, or phosphates), and isotonic agents (such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). Composition formulations may be formulated and packaged in ampoules, disposable syringes, or multi-dose vials. When needed (e.g., in injectable formulations), adequate fluidity can be maintained, for example, by using a coating (such as lecithin) or a surfactant. The absorption of antibodies or their antigen-binding fragments can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved via implants and microencapsulated delivery systems, which may include biodegradable, biocompatible polymers (e.g., ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza and Nova Pharma).

[0215] Compositions containing one or more of the antibodies or antigen-binding fragments described herein can be formulated for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) in dose units (i.e., physically discrete units containing a predetermined amount of the active compound for ease of administration and dose uniformity).

[0216] The toxicity and efficacy of the composition can be determined in cell cultures or laboratory animals (e.g., monkeys) using standard pharmaceutical methods. The LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. When an agent exhibits adverse side effects, care should be taken to minimize potential harm (i.e., reduce adverse side effects). Toxicity and efficacy can be determined using other standard pharmaceutical methods.

[0217] Data obtained from cell culture assays and animal studies can be used to formulate appropriate doses of any given agent for a subject (e.g., a human). The therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) will be the amount by which the disease is treated (e.g., to kill cancer cells), and one or more symptoms of the disease are reduced in a subject (e.g., a person identified as having cancer) or a subject identified as at risk of developing the disease (e.g., a subject who previously had cancer but is now cured). The effectiveness and dosage of any of the antibodies or antigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors can influence the dosage and timing required for effective treatment of a subject (e.g., the severity of the disease or condition, prior treatment, the subject's general health and / or age, and the presence of other diseases).

[0218] Exemplary doses include milligrams or micrograms of either the antibody or antigen-binding fragment described herein, or the antibody-drug conjugate, per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg). While these doses cover a wide range, those skilled in the art will understand that the efficacy of therapeutic agents (including antibodies and their antigen-binding fragments) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered initially, which may then be gradually increased by the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the development phase) until an appropriate response is obtained. Furthermore, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health condition, sex and diet, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in the body.

[0219] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use. This disclosure also provides methods for preparing antibodies or antigen-binding fragments thereof, or antibody-pharmaceutical conjugates, for the various uses described herein.

[0220] Example

[0221] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0222] Example 1. Generation of anti-TIGIT antibodies

[0223] Human TIGIT protein (hTIGIT-Fc, ACRO Biosystems Inc., Cat#: TIT-H5254) and cynomolgus / rhesus monkey TIGIT protein (cynTIGIT-Fc, ACRO Biosystems Inc., Cat#: TIT-C5254), or expression plasmids encoding human TIGIT or rhesus monkey TIGIT, were emulsified with adjuvants and used to immunize RenMab™ mice.

[0224] RenMab™ mice possess both humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci. The heavy chain immunoglobulin locus is a region on the chromosome containing antibody heavy chain genes. This locus includes the IGHV (variable) gene, IGHD (diversity) gene, IGHJ (connector) gene, and heavy chain constant domain gene. The κ chain immunoglobulin locus is a region on the chromosome containing antibody light chain (κ chain) genes. The κ chain immunoglobulin locus includes the IGKV (variable) gene, IGKJ (connector) gene, and light chain constant domain gene. A detailed description of RenMab™ mice can be found in PCT / CN2020 / 075698, the full text of which is incorporated herein by reference.

[0225] A total of five immunizations were administered. The first and second immunizations were spaced two weeks apart, and the remaining immunizations were spaced one week apart. One week after the fifth immunization, retro-orbital blood was collected, and serum antibody titers were measured by FACS. Boosting immunizations were administered at least fourteen days after the aforementioned immunizations (either by injecting protein or by injecting plasmids). TIGIT protein was administered intraperitoneally, and CHO-S cells expressing the human TIGIT antigen were administered via tail vein injection.

[0226] Isolate antigen-specific immune cells from immunized mice to further obtain anti-TIGIT antibodies or the light and heavy chain variable region sequences of anti-TIGIT antibodies. For example, using single-cell techniques (e.g., using Beacon). ®The Optofluidic System (Berkeley Lights Inc.) is used to screen and identify plasma cells that secrete antigen-specific monoclonal antibodies, and reverse transcription and PCR sequencing are used to obtain the variable region sequence of the antibody. The obtained variable region sequence is cloned into a vector containing the sequence encoding the human IgG1 constant region for antibody expression. The binding of the expressed antibody to TIGIT is verified by FACS. Exemplary antibodies obtained by this method include 5F1, 15C5, 36F3, and 36H7.

[0227] In addition, phage display was performed to screen for and identify antigen-specific monoclonal antibodies. 9H11 is an exemplary antibody obtained using this method.

[0228] The VH and VL CDR 1-3 sequences of 5F1, 9H11, 15C5, 36F3, and 36H7 are as follows: Figure 1 and Figure 2 As shown. The VH and VL regions of 5F1, 9H11, 15C5, 36F3, and 36H7 are as follows. Figure 3 As shown.

[0229] For comparison, the following experiments included a reference antibody labeled PC targeting TIGIT. This antibody PC has the VH / VL sequence and human IgG1 constant region as shown in SEQ ID NO: 57-58.

[0230] The constant regions of the aforementioned antibodies may also include some mutations. Regarding antibody nomenclature, when an LALA mutation (EU numbers: L234A and L235A) is introduced into the Fc region of an antibody, "LALA" is added to the antibody name. For example, if an LALA mutation is introduced into 5F1, the antibody is named 5F1-LALA.

[0231] In another experiment, the “EN” mutated to “ED” in the VH CDR of the antibody described herein to generate a CDR-modified VH. For example, in one embodiment, the “EN” mutated to “ED” in the VH CDR2 of 15C5, and the resulting CDR-modified VH was named 15C5-ND VH. The sequence of this CDR-modified VH is shown below. Figure 3 .

[0232] In another experiment, the “YS” mutated to “YG” in the VH CDR of the antibody described herein to generate a CDR-modified VH. For example, in one embodiment, the “YS” mutated to “YG” in the VH CDR2 of 15C5, and the resulting CDR-modified VH was named 15C5-SG VH. The sequence of this CDR-modified VH is shown below. Figure 3 .

[0233] Example 2. Binding affinity of anti-TIGIT antibody

[0234] Using the Biacore™ (Biacore, Inc., Piscataway NJ) 8K biosensor equipped with a pre-immobilized protein A sensor chip, surface plasmon resonance (SPR) was used to verify the binding affinity of anti-TIGIT antibodies to human His-tagged TIGIT protein (hTIGIT-His, ACRO biosystems, Cat#: TIT-H52H3) or monkey His-tagged TIGIT protein (cynTIGIT-His, ACRO biosystems, Cat#: TIT-C5223).

[0235] Purified anti-TIGIT antibody was captured on a Series S Sensor Chip Protein A for detection. 1 μg / mL or 2 μg / mL of purified anti-TIGIT antibody was loaded at a flow rate of 10 μL / min to bind to 100 nM recombinant hTIGIT-His or cynTIGIT-His. The flow rate was 30 μL / min, and the binding and dissociation times were set to 180 seconds and 400 seconds, respectively. After the last injection of each titration cycle, the chip was regenerated with glycine solution (pH 2.0) at a rate of 30 μL / min for 30 seconds.

[0236] The data were fitted to a 1:1 Langmuir fusion model using Biacore™ 8K evaluation software 3.0 (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994). Methods Enzymology (6.99-110), thus simultaneously obtaining the kinetic binding rate (kon) and dissociation rate (koff). The affinity is derived from the quotient of the kinetic rate constant (KD=koff / kon).

[0237] As will be understood by those skilled in the art, the same method was used for each test antibody, with parameters (e.g., antibody concentration) adjusted appropriately. The results for the test antibodies are summarized in the table below.

[0238] Table 1

[0239] The results showed that all anti-TIGIT antibodies could bind to human and monkey TIGIT with high affinity.

[0240] In addition, the binding affinity of 5F1 to mouse TIGIT (ACRO biosystems, Cat#: TIT-M52E6) was tested, with a KD of 6.70E-09 M.

[0241] Example 3. Blocking assay of anti-TIGIT antibody

[0242] This experiment was conducted to test whether the anti-TIGIT antibody could block the TIGIT / CD155 pathway.

[0243] CHO-S-hTIGIT cells (obtained by transfecting CHO-S cells with a vector expressing human TIGIT (SEQ ID NO: 56)) were seeded in 96-well plates (cell density 2 × 10⁶). 5 Cells / well). Anti-TIGIT antibodies 5F1, 9H11, 15C5, 36F3, 36H7, and PC were serially diluted (2-fold) to a maximum concentration of 50 μg / mL. The detection buffer was phosphate-buffered saline (PBS). 30 μL of antibody was added to each well. PBS was added to each well as a negative control. The 96-well plate was incubated at 4°C for 15 minutes. After incubation, 0.3 μg / mL of biotinylated human CD155 protein ligand (ACRObiosystems, Cat#: TIT-C5223) was added to the plate, and the plate was incubated at 4°C for 20 minutes. Then, after washing once with PBS, the cells were incubated with a 1:5000 dilution of the secondary antibody anti-hIgG-Fc-Alexa Fluor 647 (Jackson, Cat#: 109-606-170) and a 1:100 dilution of PE streptavidin at 4°C for 15 min, followed by flow cytometry analysis. Mean fluorescence intensity (MFI) was measured. A fitted curve was obtained with antibody concentration (log10, μg / mL) as the X-axis and MFI as the Y-axis. Results are as follows: Figures 4A-4C As shown.

[0244] The results showed that 5F1, 9H11, 36F3, and 36H7 blocked the interaction between TIGIT and its ligand CD155 (e.g., Figure 4A and Figure 4B As shown). On the other hand, 15C5 does not block the binding of TIGIT to CD155 (as shown). Figure 4C (As shown).

[0245] Example 4. Epitope analysis of anti-TIGIT antibody

[0246] The relative positions of target protein epitopes between paired purified anti-TIGIT antibodies were analyzed using the ForteBio Octet system via biological layer interferometry (BLI).

[0247] A 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the run buffer for the entire experiment. The ligand hTIGIT-His protein at a concentration of 5 μg / mL was captured at a flow rate of 10 µL / min, and 200 nM antibody was injected at a flow rate of 30 µL / min to bind the ligand. Another antibody was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. The binding time for each antibody was 300 seconds.

[0248] Binding values ​​for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody binding to another, binding ratios were calculated to compare each antibody pair. The binding ratio was defined as the binding value of the second antibody divided by the binding value of the first antibody. A threshold of 0.5 was used to determine whether two antibodies would interfere with each other. For example, a binding ratio greater than 0.5 indicates that the second antibody can still bind to TIGIT after the first antibody has bound. Therefore, the two antibodies may recognize different epitopes.

[0249] The results showed that 9H11, 15C5 and 36F3 identified distinct epitopes (data not shown).

[0250] Example 5. Stability analysis of anti-TIGIT antibody

[0251] The anti-TIGIT antibody was diluted to 2 mg / mL with a pH 6.0 buffer (3 mg / mL histidine, 80 mg / mL sucrose, and 0.2 mg / mL Tween 80). The diluted antibody was stored in sealed Eppendorf tubes at 40 ± 2 °C (hereinafter referred to as 40 °C) for 7 days, and its thermal stability was assessed.

[0252] Low pH stability: The stability index of the antibody was determined before and after incubation in 1 mol / L acetic acid at pH 3.5 for 6 hours.

[0253] Specifically, the following tests were performed: (1) Observing the appearance of the solution and the presence of visible insoluble matter; (2) Detecting the change in antibody purity by size exclusion high performance liquid chromatography (SEC-HPLC) (represented as the percentage of the main peak area to the total area of ​​all peaks (purity, %)); (3) Detecting the charge change of the antibody by capillary isoelectric focusing (cIEF) (represented as the percentage of the main component, acidic component and basic component); (4) Detecting the change in apparent hydrophobicity of the antibody by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (represented as the retention time of the main peak (HIC, min); (5) Detecting the change in antibody purity under non-reducing conditions (CE-SDS(NR)) by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) (represented as the percentage of the main peak area to the total area of ​​all peaks (purity, %)).

[0254] In SEC-UPLC experiments, antibody samples were diluted to 1 mg / mL with purified water and an Agilent 1290 chromatography system (connected to an XBridg protein BEH SEC column (200 Å, Waters Corporation)) was used. The following parameters were used: stationary phase: 0.1 M phosphate-buffered saline (PB) + 10% CAN, pH 7.4; flow rate: 1.8 mL / min; column temperature: 25 °C; detection wavelengths: 280 nm, 220 nm; injection volume: 10 μL; sample tray temperature: approximately 4 °C; run time: 7 minutes.

[0255] In cIEF experiments, the Maurice cIEF Method Development Kit (Protein Simple, Cat#: PS-MDK01-C) was used for sample preparation. Specifically, 40 μg of protein sample was mixed with the following reagents from the kit: 1 μL Maurice cIEF pI label -4.05, 1 μL Maurice cIEF pI label -9.99, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500mM arginine, 4 μL amphoteric electrolyte (Pharmalyte pH range 3–10), and water (added to bring the final volume to 100 μL). Imaging capillary isoelectric focusing spectra were generated on a Maurice analyzer (Protein Simple, Santa Clara, CA) using the Maurice cIEF cartridge (PS-MC02-C). The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to analyze the absorbance of the protein focused at 280 nm.

[0256] In the HIC-HPLC experiments, an Agilent 1260 chromatographic system (connected to a ProPa HIC-10 column (4.6 × 250 mm, Thermo Scientific)) was used, and the sample was diluted 10-fold using mobile phase A. The following parameters were used: Mobile phase A: 0.9 M ammonium sulfate, 0.1 M phosphate buffer (PB), 10% acetonitrile, pH 6.5; Mobile phase B: 0.1 M phosphate buffer (PB), 10% acetonitrile, pH 6.5; Flow rate: 0.8 mL / min; Gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; Column temperature: 30 °C; Detection wavelength: 280 nm, 220 nm; Injection volume: 10 μg; Sample tray temperature: approximately 10 °C; Run time: 50 min.

[0257] In the CE-SDS (NR) experiment, Maurice (Protein simple, Maurice) was used. ™ The sample was prepared using the Maurice CE-SDS Size Application Kit (Protein Simple, Cat#: PS-MAK02-S). 54 μL of sample buffer, 6 μL of antibody sample, 2.4 μL of 25× internal standard, and 3 μL of 250 mM iodoacetamide (SIGMA, Cat#: 16125) were added to a microcentrifuge tube, followed by centrifugation at 3000 rpm for 1 minute and heating in a 70°C water bath for 10 minutes. The sample was then cooled to room temperature and centrifuged at 10000 rpm for 3 minutes. The supernatant was then transferred to a 96-well plate and tested in Maurice. The following parameters were used: injection voltage 4.6 kV, injection time 20 seconds, separation voltage 5.75 kV, and separation time 40 minutes.

[0258] Detailed results for 5F1 and 9H11 are shown in the table below. The results indicate that these anti-TIGIT antibodies possess good stability and physicochemical properties.

[0259] Table 2

[0260] Note: - Not detected.

[0261] Example 6. Pharmacokinetic (PK) Analysis

[0262] The pharmacokinetic properties of the anti-TIGIT antibody were determined in C57BL / 6 mice. Specifically, mice were divided into four groups (n=3 per group) and administered 3 mg / kg of 9H11, 15C5-SG, 36F3, or PC via intravenous injection. Blood samples were collected 4 days before administration and at 15 minutes, 4 hours, 1 day, 2 days, 4 days, and 6 days after administration.

[0263] Serum levels of human antibodies were determined using a sandwich ELISA. In short, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., Cat#: 109-005-088) was diluted to a final concentration of 2000 ng / mL, and 100 μL / well was added to a 96-well ELISA plate, which was then incubated overnight at 4°C. After incubation, the antibody levels were measured with PBS-T buffer (PBS with added Tween). TM 20) Wash 4 times. Block unbound areas with 2% BSA (bovine serum albumin) at 37°C for 1 hour. Then, wash the plate 4 times with PBS-T buffer. After washing, add 100 μL of blocking buffer (2% BSA) to each well. Seal the wells and incubate at 37°C for 1 hour. After washing the plate with a plate washer, add 100 μL / well of peroxidase AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch Inc., Cat#: 109-036-098) to each well and incubate at 37°C for 1 hour. After washing, add 100 μL / well of tetramethylbenzidine (TMB) solution as substrate to each well of a 96-well plate. After incubating at room temperature in the dark, add 100 μL of stop solution (Beyotime, Cat#: P0215) to each well. The absorbance values ​​of each well were read using a microplate reader at wavelengths of 450 nm and 630 nm. Data analysis was performed using Gen5 software. TM The data was analyzed. Using the absorbance values ​​and corresponding concentrations of calibration samples prepared for each test product, four parameters (i.e., T) were plotted. 1 / 2 C max AUC 0-∞ (and CL) standard curve.

[0264] Antibody concentrations in each serum sample were calculated using a standard curve. Drug concentration-time curves were plotted using the calculated sample concentrations at each time point. Phoenix was used. TM WinNolin 8.3 calculates pharmacokinetic parameters.

[0265] like Figure 5As shown, the results are consistent with typical pharmacokinetic characteristics, indicating that the antibody concentration in the serum of C57BL / 6 mice decreased over time after injection of anti-TIGIT antibody.

[0266] Example 7. ADCC Activity Assay

[0267] Experiments were conducted to test the ADCC (antibody-dependent cytotoxicity) activity of the anti-TIGIT antibody. Specifically, effector cells Jurkat-luc-hCD16A (obtained by transfecting Jurkat cells with a vector expressing human CD16A (SEQ ID NO: 60)) and target cells CHO-S-TIGIT (obtained by transfecting CHO-S cells with a vector expressing human TIGIT (SEQ ID NO: 56)) were co-incubated with serially diluted anti-TIGIT antibody at an E:T ratio of 4:1 for 6 hours. After incubation, 75 μL of luciferase substrate was added, and the mixture was incubated at room temperature for 5 minutes. Then, the mixture was placed in a 96-well microplate chemiluminescence assay to detect cell-killing activity. EC50 was calculated.

[0268] The results are shown in Table 3 and Figure 6 As shown, all tested anti-TIGIT antibodies exhibited ADCC effects, and the four anti-TIGIT antibodies 9H11, 15C5-SG, 36F3, and 36H7 had higher ADCC activity than the positive control PC.

[0269] Table 3

[0270] Example 8. Antitumor activity of anti-TIGIT antibody in hTIGIT model

[0271] A humanized TIGIT mouse model (hTIGIT mouse) was engineered to express the chimeric TIGIT protein (SEQ ID NO:59), wherein a portion of the extracellular region of the mouse TIGIT protein was replaced by the corresponding extracellular region of the human TIGIT protein. A detailed description of the humanized TIGIT mouse model can be found in PCT / CN2017 / 099576, the full text of which is incorporated herein by reference.

[0272] Approximately 5×10 5 MC38 cells were subcutaneously injected into hTIGIT mice. When the tumor volume reached approximately 100-150 mm³, the mice were divided into a control group and four treatment groups (n=6 per group) based on tumor size. Treatment groups were randomly assigned to receive 9H11, 36F3, 36H7, or PC treatment. Control group mice were injected with phosphate-buffered saline (PBS). Tumor volume was measured twice weekly, and the mice were weighed. Mice were euthanized when the tumor volume reached 3000 mm³. Details of the dosing regimen are shown in the table below.

[0273] Table 4

[0274] The long and short axis lengths of the tumors were measured, and the tumor volume was calculated as 0.5 × long axis × (short axis)². Mouse body weight was measured twice weekly. The tumor growth inhibition percentage (TGI%) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero. Statistical analysis was performed using the t-test. P < 0.05 was the threshold for statistical significance.

[0275] Mice in all groups showed an increase in body weight. On the day of grouping (day 0), the average body weight of each group ranged from 21.8 g to 22.1 g; at the end of the experiment (day 21), the average body weight of each group ranged from 23.9 g to 24.8 g. Therefore, the change in average body weight for each group ranged from 109.4% to 112.4%. The results indicate that the tested antibody was well tolerated and had no significant toxicity to mice.

[0276] Tumor size in the antibody treatment group was as follows Figure 7 As shown in the table below, the results of this experiment are summarized, including tumor volume, mouse survival rate, tumor growth inhibition value (TGI%), and statistical differences (P-value) in tumor volume and body weight between the treatment group and the control group on the day of grouping (day 0), 11 days after grouping (day 11), and at the end of the experiment (day 21).

[0277] Table 5

[0278] like Figure 7 As shown in Table 5, tumor growth was inhibited to varying degrees in the treatment groups (G2 to G5) compared to the control group (G1). In the treatment groups, 36F3, 36H7, and 9H11 showed better tumor suppression than the positive control PC.

[0279] In a similar experiment, approximately 5 × 10 5 MC38 cells were subcutaneously injected into hTIGIT mice. When the tumor volume grew to approximately 100-150 mm³, the mice were divided into different groups (n=6 per group) according to tumor size. The treatment group was randomly assigned to receive 9H11, 36F3, or PC treatment. The control group mice were injected with PBS. Tumor volume and mouse weight were measured twice weekly. Mice were euthanized when the tumor volume reached 3000 mm³. Details of the dosing regimen are shown in the table below.

[0280] Table 6

[0281] Mice in all groups showed an increase in body weight. On day 0 (the day of grouping), the average body weight of each group ranged from 21.1 g to 21.5 g; on day 21, the average body weight of each group ranged from 23.1 g to 24.5 g. Therefore, the average body weight change of each group was within the range of 107.9% to 112.9%. The results indicate that the tested antibody is well-tolerated and has no significant toxicity to mice.

[0282] Tumor size in the antibody treatment group was as follows Figure 8 As shown in the table below, the results of this experiment are summarized, including tumor volume, mouse survival rate, TGI% on days 0, 11, and 21, and the p-values ​​for tumor volume and body weight between the treatment and control groups on day 21.

[0283] Table 7

[0284] like Figure 8 As shown in the table above, tumor growth was inhibited to varying degrees in the treatment groups (G2-G6) compared to the control group (G1). The TGI% of 9H11 and 36F3 at dose levels of 3 mg / kg and 10 mg / kg were both higher than the TGI% of the positive control PC at 10 mg / kg.

[0285] Example 9. Antitumor activity of anti-TIGIT antibody in hTIGIT model

[0286] Approximately 5×10 5 MC38 cells were subcutaneously injected into hTIGIT mice. When the tumor volume reached approximately 100-150 mm³, the mice were divided into a control group and two treatment groups (n=5 per group) based on tumor size. Treatment groups were randomly assigned to receive either 15C5 or PC treatment. Control group mice were injected with PBS. Tumor volume and mouse weight were measured twice weekly. Mice were euthanized when the tumor volume reached 3000 mm³. Details of the dosing regimen are shown in the table below.

[0287] Table 8

[0288] Mice in all groups showed an increase in body weight. On the day of grouping (day 0), the average body weight of each group was 19.9 g. On day 21, the average body weight of each group ranged from 22.7 g to 23.8 g. The change in average body weight among the groups ranged from 114.0% to 120.0%. The results indicate that the tested antibody was well tolerated and had no significant toxicity to mice.

[0289] Tumor size in the antibody treatment group was as follows Figure 9 As shown in the table below, the results of this experiment are summarized, including tumor volume, mouse survival rate, TGI% on days 0, 11, and 21, and the p-values ​​for tumor volume and body weight between the treatment and control groups.

[0290] Table 9

[0291] like Figure 9 As shown in Table 9, 15C5 exhibited better tumor suppression compared to the positive control PC.

[0292] Other implementation plans

[0293] It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to TIGIT (a T-cell immune receptor having Ig and ITIM domains), comprising: The heavy chain variable regions (VHs) include complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region contains an amino acid sequence having at least 80% identity with a selected VH CDR1 amino acid sequence, the VH CDR2 region contains an amino acid sequence having at least 80% identity with a selected VHCDR2 amino acid sequence, and the VH CDR3 region contains an amino acid sequence having at least 80% identity with a selected VHCDR3 amino acid sequence; and The system includes light chain variable regions (VLs) comprising CDR1, 2, and 3, wherein the VL CDR1 region contains an amino acid sequence having at least 80% identity with a selected VL CDR1 amino acid sequence, the VL CDR2 region contains an amino acid sequence having at least 80% identity with a selected VL CDR2 amino acid sequence, and the VL CDR3 region contains an amino acid sequence having at least 80% identity with a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, 3 amino acid sequences and the selected VL CDR1, 2, 3 amino acid sequences are one of the following: (1) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 1-3, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; (2) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 7-9, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, respectively; (3) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13-15, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (4) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 61, and 15, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (5) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 13, 62, and 15, respectively, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 19-21, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22-24, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 25-27, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28-30, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 31-33, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 4-6, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 34-36, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 10-12, respectively; (10) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 37-39, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 16-18, respectively; (11) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 40-42, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 22-24, respectively; and (12) The selected VH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 43-45, and the selected VLCDR1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 28-30.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 1-3, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 4-6, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 7-9, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 10-12.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 13-15, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 16-18.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 13, 61, 15, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 16-18, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 13, 62, 15, respectively, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 16-18, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 19-21, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 22-24.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat definition, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 25-27, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 28-30.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 31-33, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 4-6, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 34-36, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 10-12.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 37-39, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 16-18.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 40-42, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 22-24.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the definition of Chothia, the VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 43-45, and the VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NO: 28-30.

14. An antibody or antigen-binding fragment thereof that binds to TIGIT, comprising a heavy chain variable region (VH) containing an amino acid sequence having at least 90% identity with a selected VH sequence; and a light chain variable region (VL) containing an amino acid sequence having at least 90% identity with a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO: 47; (2) The selected VH sequence is SEQ ID NO: 48 or 65, and the selected VL sequence is SEQ ID NO: 49; (3) The selected VH sequence is SEQ ID NO: 50, and the selected VL sequence is SEQ ID NO: 51; (4) The selected VH sequence is SEQ ID NO: 63, and the selected VL sequence is SEQ ID NO: 51; (5) The selected VH sequence is SEQ ID NO: 64, and the selected VL sequence is SEQ ID NO: 51; (6) The selected VH sequence is SEQ ID NO: 52, and the selected VL sequence is SEQ ID NO: 53; and (7) The selected VH sequence is SEQ ID NO: 54, and the selected VL sequence is SEQ ID NO:

55.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 46 and the VL comprises the sequence of SEQ ID NO:

47.

16. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 48 or 65, and the VL comprises the sequence of SEQ ID NO:

49.

17. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 50, and the VL comprises the sequence of SEQ ID NO:

51.

18. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 63 and the VL comprises the sequence of SEQ ID NO:

51.

19. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 64 and the VL comprises the sequence of SEQ ID NO:

51.

20. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 52 and the VL comprises the sequence of SEQ ID NO:

53.

21. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the sequence of SEQ ID NO: 54, and the VL comprises the sequence of SEQ ID NO:

55.

22. An antibody or antigen-binding fragment thereof that binds to TIGIT, comprising: The heavy chain variable region (VH) includes VH CDR1, VH CDR2, and VH CDR3 that are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and the light chain variable region (VL) includes VL CDR1, VL CDR2, and VL CDR3 that are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) The selected VH sequence is SEQ ID NO: 46, and the selected VL sequence is SEQ ID NO: 47; (2) The selected VH sequence is SEQ ID NO: 48 or 65, and the selected VL sequence is SEQ ID NO: 49; (3) The selected VH sequence is SEQ ID NO: 50, and the selected VL sequence is SEQ ID NO: 51; (4) The selected VH sequence is SEQ ID NO: 63, and the selected VL sequence is SEQ ID NO: 51; (5) The selected VH sequence is SEQ ID NO: 64, and the selected VL sequence is SEQ ID NO: 51; (6) The selected VH sequence is SEQ ID NO: 52, and the selected VL sequence is SEQ ID NO: 53; and (7) The selected VH sequence is SEQ ID NO: 54, and the selected VL sequence is SEQ ID NO:

55.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof specifically binds to human or monkey TIGIT.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof is a human or humanized antibody or antigen-binding fragment thereof.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1-24, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

26. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof cross-competing with the antibody or antigen-binding fragment thereof according to any one of claims 1-25.

27. A nucleic acid comprising a polynucleotide encoding a polypeptide, said polypeptide comprising: (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 1-3, respectively, and wherein the VH binds TIGIT when paired with a light chain variable region (VL) comprising an amino acid sequence as shown in SEQ ID NO:

47. (2) An immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL), wherein the VL comprises complementarity-determining regions (CDRs) 1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 4-6, respectively, and wherein the VL binds TIGIT when paired with a heavy chain variable region (VH) comprising an amino acid sequence as shown in SEQ ID NO:

46. (3) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 7-9, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 49; (4) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 10-12, respectively, and wherein the VL binds TIGIT when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO: 48 or 65. (5) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13-15, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (6) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16-18, and wherein the VL binds TIGIT when paired with VH comprising an amino acid sequence as shown in SEQ ID NO: 50; (7) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13, 61 and 15 respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (8) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16-18, and wherein the VL binds TIGIT when paired with VH comprising an amino acid sequence as shown in SEQ ID NO: 63; (9) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 13, 62 and 15, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (10) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 16-18, and wherein the VL binds TIGIT when paired with a VH comprising an amino acid sequence as shown in SEQ ID NO: 64; (11) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 19-21, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 53; (12) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 22-24, respectively, and wherein the VL binds TIGIT when paired with VH comprising an amino acid sequence as shown in SEQ ID NO: 52; (13) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 25-27, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 55; (14) An immunoglobulin light chain or fragment thereof comprising VL, wherein the VL comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 28-30, respectively, and wherein the VL binds TIGIT when paired with VH comprising an amino acid sequence as shown in SEQ ID NO: 54; (15) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 31-33, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 47; (16) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 34-36, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 49; (17) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 37-39, respectively, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO: 51; (18) An immunoglobulin heavy chain or fragment thereof comprising VH, said VH comprising CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 40-42, respectively, and said VH binding TIGIT upon pairing with VL comprising an amino acid sequence as shown in SEQ ID NO: 53; and (19) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises CDR1, 2 and 3 having amino acid sequences as shown in SEQ ID NO: 43-45, and wherein the VH binds TIGIT when paired with a VL comprising an amino acid sequence as shown in SEQ ID NO:

55.

28. The nucleic acid of claim 27, wherein the VH specifically binds to human or monkey TIGIT when paired with VL.

29. The nucleic acid according to claim 27 or 28, wherein the immunoglobulin heavy chain or a fragment thereof is a human or humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or a fragment thereof is a human or humanized immunoglobulin light chain or a fragment thereof.

30. The nucleic acid according to any one of claims 27-29, wherein the nucleic acid encodes a single-stranded variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

31. The nucleic acid according to any one of claims 27-30, wherein the nucleic acid is cDNA.

32. A vector comprising one or more nucleic acids as described in any one of claims 27 to 31.

33. A vector comprising two of the nucleic acids of any one of claims 27 to 31, wherein the vector encodes a VH region and a VL region, the VH region and the VL region jointly binding TIGIT.

34. A pair of vectors, wherein each vector comprises a nucleic acid according to any one of claims 27 to 31, wherein the pair of vectors co-encode a VH region and a VL region, the VH region and the VL region co-binding TIGIT.

35. A cell comprising the vector of claim 32 or 33 or a pair of vectors of claim 34.

36. The cell of claim 35, wherein the cell is a CHO cell.

37. A cell comprising one or more nucleic acids as claimed in any one of claims 27 to 31.

38. A method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) The cells are cultured under conditions sufficient to cause the cells of any one of claims 35 to 37 to produce the antibody or an antigen-binding fragment thereof; as well as (b) Collect antibodies or antigen-binding fragments thereof produced by the cells.

39. An antibody-drug conjugate (ADC) comprising a therapeutic agent covalently bound to an antibody or an antigen-binding fragment thereof as described in any one of claims 1-26.

40. The antibody-drug conjugate of claim 39, wherein the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

41. A method for treating a subject suffering from cancer, the method comprising: The subject is given a therapeutically effective amount of the composition, the composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-26 or the antibody-drug conjugate as described in claims 39 or 40.

42. The method of claim 41, wherein the cancer is melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colon cancer, hepatocellular carcinoma (HCC), gastric cancer, breast cancer, pancreatic cancer, glioblastoma, hematologic malignancy, kidney cancer, or ovarian cancer.

43. The method of claim 41 or 42, wherein the method further comprises administering to the subject a therapeutically effective amount of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-OX40 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, an anti-4-1BB antibody, and / or an anti-CD40 antibody.

44. The method according to any one of claims 41-43, wherein the method further comprises administering chemotherapy to the subject.

45. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-26, or an antibody-drug conjugate as described in claim 39 or 40.

46. ​​A method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-26, or an antibody-drug conjugate as described in claim 39 or 40.

47. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) The antibody or antigen-binding fragment thereof as described in any one of claims 1-26; or (b) The antibody-drug conjugate of claim 39 or 40.

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