Anti-tigit antibodies and uses thereof

Abstract

The present disclosure provides anti-TIGIT polypeptides or fragments thereof. Further provided are methods of using the antibodies or fragments thereof for the treatment and diagnosis of diseases, such as cancer, infection, or immune disorders.

Description

Technical Field

[0001] This disclosure relates to anti-TIGIT peptides, including anti-TIGIT antibodies or immunologically active fragments thereof, isolated nucleic acids encoding anti-TIGIT antibodies or immunologically active fragments thereof, and their uses, particularly in treating medical diseases in which pathogenic cells utilize TIGIT / PVR checkpoints for immune evasion. This application particularly relates to humanized anti-TIGIT antibodies and their antigen-binding fragments, which can enhance the activation of the immune system against diseased tissues, including cancer cells expressing TIGIT ligands, especially PVR. Background Technology

[0002] In recent years, the immune checkpoint protein TIGIT has become a hot topic in cancer immunotherapy research and development. The binding of TIGIT to its homologous ligand PVR (poliovirus receptor, or CD155) is an important mechanism for tumor immune escape, directly inhibiting lymphocyte activation. The role of TIGIT / PVR in tumor immune surveillance is similar to that of the PD-1 / PD-L1 axis in tumor immunosuppression. Both TIGIT and PD-1 are upregulated in various cancers. Now, TIGIT / PVR has emerged as a new immune checkpoint following PD-1 / PD-L1.

[0003] TIGIT is highly expressed on the surface of many types of lymphocytes, especially tumor-infiltrating lymphocytes. These lymphocytes include effector CD4+ T cells, regulatory CD4+ T cells, effector CD8+ T cells, and NK cells. Effector T cells are the main force in killing tumors. They are primarily generated by stem cell-like memory T cells. Stem cell-like memory T cells express PD-1 and TIGIT, but do not express other negative regulatory factors (such as Tim-3), allowing the combined use of TIGIT inhibitors or antibodies with PD-1 / PD-L1 inhibitors to activate stem cell-like memory T cells to continuously generate effector T cells, thereby exerting a synergistic anti-tumor effect. At the ASCO 2020 meeting, clinical data on the treatment of non-small cell lung cancer (NSCLC) with the anti-TIGIT monoclonal antibody tiragolumab in combination with the anti-PD-1 monoclonal antibody atezolizumab were announced. These results are encouraging, showing that this therapy has the potential to challenge first-line treatment for NSCLC. Furthermore, in current clinical trials, the TIGIT antibody is also being used in combination with daratumumab (targeting CD38) / rituximab (targeting CD20) for the treatment of multiple myeloma / B-cell non-Hodgkin lymphoma, and with pomalidimide.

[0004] (Cereblon ligand) is used in combination with chemotherapy to treat multiple myeloma, in combination with pembrolizumab (anti-PD-1) and CTLA-4 inhibitor / lenvatinib (tyrosine kinase inhibitor) to treat melanoma, and in combination with zimberelimab (anti-PD-1) and AB928 (biadenosine receptor antagonist) to treat non-small cell lung cancer.

[0005] On June 18, 2018, a research paper titled "Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity" (NatImmunol.2018Jul;19(7):723-732.doi:10.1038 / s41590-018-0132-0.Epub 2018 Jun 18) revealed that the inhibitory receptor TIGIT can lead to NK cell exhaustion during tumor development, and demonstrated that anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and be used in immunotherapy for various tumors.

[0006] Currently, proof-of-concept trials of TIGIT inhibitors have been completed, with encouraging results regarding their safety and efficacy. Many domestic and international pharmaceutical companies have invested in their research and development. However, as a therapeutic agent, anti-TIGIT antibodies still have room for improvement. Therefore, there is a need in this field to develop novel anti-TIGIT antibodies with higher specificity and efficacy. Summary of the Invention

[0007] This article presents an antibody and its immunomodulatory fragment that binds with high affinity to TIGIT molecules expressed on cells (e.g., cancer cells) and promotes an effective immune response against cancer cells. The antibody and its immunomodulatory fragment provided in this article can enhance the activation of the immune system, thus providing important therapeutic and diagnostic agents for targeting pathological conditions associated with the expression and / or activity of TIGIT molecules. In one aspect, this disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to the extracellular domain of TIGIT with a binding affinity better than or about 10 nM, better than 8 nM or about 8 nM, better than 6 nM or about 6 nM, better than 4 nM or about 4 nM, better than 2 nM or about 2 nM, better than 1 nM or about 1 nM, better than 0.8 nM or about 0.8 nM, better than 0.6 nM or about 0.6 nM, better than 0.4 nM or about 0.4 nM, better than 0.2 or about 0.2 nM, as determined by SPR analysis, for example about 0.1-0.2 nM, about 0.1-0.18 nM, about 0.1-0.13 nM, about 0.1 nM, 0.11 nM, 0.12 nM, or better, as determined by SPR analysis.

[0008] In some embodiments, this disclosure provides an antibody or an antigen-binding fragment thereof comprising at least one of the following:

[0009] (a) A CDR1H sequence containing GYTFSRYWIE (SEQ ID NO:1),

[0010] (b) A CDR2H sequence containing EIFPGSGGTNYNEKFKG (SEQ ID NO:2),

[0011] (c) A CDR3H sequence containing HLGALDY (SEQ ID NO:3),

[0012] (d) A CDR1L sequence containing SASSVSYIH (SEQ ID NO:4),

[0013] (e) A CDR2L sequence containing RTSNLAS (SEQ ID NO:5), and

[0014] (f) A CDR3L sequence containing QQYHSNPWT (SEQ ID NO:6).

[0015] In some embodiments, this application provides an antibody or an antigen-binding fragment thereof, wherein

[0016] (a) The HC contains

[0017] The CDR1H sequence containing GYTFSRYWIE (SEQ ID NO:1)

[0018] The CDR2H sequence containing EIFPGSGGTNYNEKFKG (SEQ ID NO:2), and

[0019] The CDR3H sequence containing HLGALDY (SEQ ID NO:3)

[0020] (b) The LC includes

[0021] The CDR1L sequence containing SASSVSYIH (SEQ ID NO:4)

[0022] The CDR2L sequence containing RTSNLAS (SEQ ID NO:5), and

[0023] The CDR3L sequence containing QQYHSNPWT (SEQ ID NO:6).

[0024] The CDR sequence was determined according to Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols.1-3.

[0025] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody or its antigen-binding fragment of the present disclosure further comprises a human receptor framework. In some embodiments, the human receptor framework is derived from a human immunoglobulin framework or a human common framework. In some embodiments, the human receptor framework comprises a VL isotype κI framework sequence and a VH isotype III framework sequence. Generally, the isotypes of the sequences are as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In some embodiments, for VL, the isotype is isotype κI as described in Kabat et al. (ibid.). In some embodiments, for VH, the isotype is isotype III as described in Kabat et al. (ibid.).

[0026] In some embodiments, the antibody or its antigen-binding fragment comprises a human common framework. In some embodiments, the antibody or its antigen-binding fragment comprises a human common framework with changes in the amino acid sequence, such as changes of 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acids.

[0027] In some embodiments, the antibody or antigen-binding fragment of the present disclosure comprises an HC variable region sequence containing the amino acid sequence shown in SEQ ID NO:7, 8, or 9, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7, 8, or 99. In some embodiments, the antibody or antigen-binding fragment of the present disclosure comprises an LC variable region sequence containing the amino acid sequence shown in SEQ ID NO:10, 11, or 12, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:10, 11, or 12. In some embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. In some embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:9, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:10.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure comprises an HC sequence containing the amino acid sequence shown in SEQ ID NO:13, 14, or 15, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:13, 14, or 15. In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure comprises an LC sequence containing the amino acid sequence shown in SEQ ID NO:16, 17, or 18, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:16, 17, or 18. In some embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO:14, and the LC sequence comprises the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO:13, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO:15, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:16.

[0029] In some embodiments, the antibody is an IgG1, IgG2, or IgG4 isotype. In some embodiments, the antigen-binding fragment is selected from any one or more of the following: Fab, F(ab')2, Fab', scFv, and Fv. In some embodiments, the antibody or antigen-binding fragment of this disclosure is a blocking antibody or antagonist antibody that inhibits or reduces the biological activity of the TIGIT molecule it binds to. Preferably, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the TIGIT molecule.

[0030] In one aspect, this disclosure provides a bispecific antibody comprising an antibody of the present disclosure or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof. In some embodiments, the second antibody or its antigen-binding fragment specifically binds to a tumor antigen expressed on the surface of tumor cells, wherein the tumor antigen is selected from any one or more of the following: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R; EphA 2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; Human papillomavirus-E6; Human papillomavirus-E7; JAM-3; KID3; KID31; KSA(17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosyltransferase; Tumorin M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; Transferrin receptor; and VEGF receptor. In some embodiments, the second antibody or its antigen-binding fragment specifically binds to checkpoint proteins expressed on the surface of abnormal cells or immune cells, wherein the immune checkpoint proteins are selected from any one or more of the following: 2B4; 4-1BB; 4-1BB ligand; B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; galactoglobulin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; VSIG8.

[0031] In one aspect, this disclosure provides a polypeptide comprising the antibody or antigen-binding fragment thereof disclosed herein.

[0032] In one aspect, this disclosure provides a polypeptide comprising the HC variable region and / or LC variable region of an antibody or an antigen-binding fragment thereof disclosed herein.

[0033] In one aspect, this disclosure provides conjugates comprising the antibodies of this disclosure or antigen-binding fragments thereof. In some embodiments, this disclosure provides conjugates consisting of the antibodies of this disclosure or antigen-binding fragments thereof, linked to a therapeutic agent. In some embodiments, the therapeutic agent is an immunomodulator. In some embodiments, the therapeutic agent is an immunomodulator. In some embodiments, the therapeutic agent is a cytotoxin or a radioisotope.

[0034] In one aspect, this disclosure provides a composition comprising an antibody or antigen-binding fragment thereof, a bispecific antibody, a peptide, a conjugate, and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises an anticancer agent. In some embodiments, the agent is an antibody, a chemotherapeutic agent, a radiotherapy agent, a hormone therapy agent, a toxin, or an immunotherapy agent. In some embodiments, the composition further comprises an antibody or agent that inhibits checkpoints.

[0035] In one aspect, this disclosure provides an article or kit for treating cancer, comprising an antibody or antigen-binding fragment thereof of the present disclosure, a bispecific antibody, a polypeptide, a conjugate or composition thereof, and a packaging insert containing necessary information regarding the use of the antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate or composition relating to the present disclosure.

[0036] In one aspect, this disclosure provides an article or kit for diagnosing cancer or determining the presence and / or amount of TIGIT, comprising an antibody of this disclosure or an antigen-binding fragment thereof, and a packaging insert containing necessary information regarding the use of the antibody of this disclosure or an antigen-binding fragment thereof.

[0037] In one aspect, this disclosure provides isolated nucleic acids encoding antibodies or antigen-binding fragments thereof of the present disclosure. In some embodiments, this disclosure provides isolated nucleic acids encoding HC variable regions and / or LC variable regions of antibodies or antigen-binding fragments thereof of the present disclosure. In some embodiments, this disclosure provides expression vectors comprising said nucleic acids, or host cells comprising said expression vectors.

[0038] In one aspect, this disclosure provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising expressing the antibody or an antigen-binding fragment thereof in the host cells and isolating the antibody or an antigen-binding fragment thereof from the host cells.

[0039] In one aspect, this disclosure provides a method for treating cancer, comprising administering to a patient suffering from cancer an effective amount of the antibody or antigen-binding fragment thereof of this disclosure, a bispecific antibody, a polypeptide, a conjugate, a composition, an article, or a kit. In some embodiments, the cancer is selected from one or more of the following: lymphoma, melanoma, colorectal adenocarcinoma, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, gastric cancer, and clear cell renal cell carcinoma. In some embodiments, the cancer originates from a solid tumor.

[0040] In one embodiment, an effective amount of the antibodies or antigen-binding fragments, bispecific antibodies, peptides, conjugates, compositions, articles, or kits of the present disclosure is the sole therapeutic anticancer agent administered to a patient. In another embodiment, they may be administered in combination with another antibody or antibody fragment or anticancer agent, said antibody or antibody fragment or anticancer agent including, but not limited to, antibodies against checkpoint molecules or their receptors (e.g., anti-CTLA-4 antibody, anti-B7S1 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-B7H3 antibody, etc.); anti-epidermal growth factor receptor (EGFR) agents, such as panitumumab, and the anti-EGFR antibody cetuximab. ), and the EGFR tyrosine kinase (TK) inhibitor gefitinib. ) and erlotinib Alkylating agents, such as cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, nitrogen mustard, cyclophosphamide, chlorambucil, and ifosfamide; paclitaxel and docetaxel; and topoisomerase inhibitors, such as, for example, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide.

[0041] In some embodiments, the antibodies or antigen-binding fragments thereof, bispecific antibodies, peptides, conjugates, compositions, articles or kits of the present disclosure as described above are administered in combination with anti-PD-1 antibodies or anti-PD-L1 antibodies to achieve a synergistic effect in cancer treatment.

[0042] In one aspect, this disclosure provides a method of treating cancer, comprising administering to a subject with cancer an effective amount of an antibody of the present disclosure or an antigen-binding fragment thereof, a bispecific antibody, a polypeptide, a conjugate, a composition, an article, or a kit. In some embodiments, the cancer is selected from one or more of the following: prostate cancer, colon cancer, gastric cancer, clear cell renal cell carcinoma, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is selected from one or more of the following: colorectal cancer with high microsatellite instability, colorectal cancer with stable microsatellite activity, triple-negative breast cancer, Merkel cell carcinoma, endometrial cancer, and esophageal cancer.

[0043] In one aspect, this disclosure provides a method for treating cancer, comprising: a) treating T cells and / or NK cells in vitro with the antibodies described above or antigen-binding fragments thereof, bispecific antibodies, peptides, conjugates, compositions, articles of manufacture, or kits of this disclosure; and b) administering the treated T cells and / or NK cells to a patient. In some embodiments, the method further comprises isolating T cells and / or NK cells from an individual prior to step a). In some embodiments, the T cells and / or NK cells are derived from a patient to be treated. In some embodiments, the T cells are tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0044] Therefore, in one aspect, this disclosure also provides lymphocytes, such as T cells or NK cells, derived from a subject and treated in vitro with the antibodies or antigen-binding fragments thereof described in this application, bispecific antibodies, peptides, conjugates, compositions, articles, or kits. In some embodiments, the T cells and / or NK cells are derived from a patient to be treated. In some embodiments, the T cells are tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0045] In one aspect, this disclosure provides a method for treating or suppressing an infection in a patient in need, comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof of this disclosure, a bispecific antibody, a polypeptide, a conjugate, a composition, an article, or a kit. In some embodiments, the infection is a viral, bacterial, fungal, or parasitic infection. In certain specific embodiments, the infection is an HIV infection.

[0046] In one aspect, this disclosure provides a method for detecting or quantifying the expression or activity of a TIGIT peptide, comprising contacting an antibody or antigen-binding fragment of the present disclosure with a sample from a subject. In some embodiments, the antibody or antigen-binding fragment is labeled with a detectable substance. In some embodiments, the antibody or antigen-binding fragment is radiolabeled, fluorescently labeled, or enzyme-labeled.

[0047] In one aspect, this disclosure provides a method for predicting the risk of cancer in a subject, the method comprising detecting, quantifying, or monitoring the expression or activity of a TIGIT peptide by using an antibody or antigen-binding fragment thereof disclosed herein.

[0048] In one aspect, this disclosure provides a method for monitoring the effectiveness of a drug in treating cancer, said cancer showing an increase in TIGIT expression or activity, the method comprising detecting or quantifying the expression or activity of a TIGIT peptide by using an antibody or antigen-binding fragment thereof disclosed herein.

[0049] In one embodiment, this disclosure provides an isolated polynucleotide encoding the aforementioned human anti-TIGIT antibody or a fragment thereof.

[0050] In one embodiment, this disclosure provides a method for diagnosing diseases, dysregulations, or conditions associated with TIGIT expression on cells, or for determining the presence and / or amount of TIGIT, said method comprising a) contacting cells with a human anti-TIGIT antibody or a fragment thereof, said antibody or fragment thereof comprising a subset selected from SEQ ID NO:7-18; and b) detecting the presence of TIGIT, said presence of TIGIT being diagnostic of diseases, dysregulations, or conditions associated with TIGIT expression. In some embodiments, the disease, dysregulation, or condition associated with TIGIT expression is cancer.

[0051] In one embodiment, this disclosure provides a method for diagnosing, prognosing, or determining the risk of TIGIT-related disease in mammals, wherein the method includes detecting TIGIT expression in a sample from a mammal, comprising a) contacting the sample with a human anti-TIGIT antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO:7-18; and b) detecting the presence of TIGIT, wherein the presence of TIGIT is diagnostic of a TIGIT-related disease in mammals. In some embodiments, the TIGIT-related disease is cancer.

[0052] In one embodiment, this disclosure provides a method for inhibiting TIGIT-dependent T cell and / or NK cell suppression, wherein the method includes contacting cells with a human anti-TIGIT antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO:7-18. In some embodiments, the cells are selected from TIGIT-expressing lymphocytes, such as effector CD4+ T cells, regulatory CD4+ T cells, effector CD8+ T cells, or NK cells.

[0053] In one embodiment, this disclosure provides a method for blocking TIGIT-dependent immunosuppression in mammals, wherein the method comprises administering an effective amount of the aforementioned anti-TIGIT antibody or a fragment thereof to the mammal. In some embodiments, the mammal comprises cells selected from any of the following: TIGIT-expressing lymphocytes (e.g., effector CD4+ T cells, regulatory CD4+ T cells, effector CD8+ T cells, or NK cells) and abnormal cells expressing PVR, PVRL2, and / or PVRL3.

[0054] In one embodiment, this disclosure provides a method for providing antitumor immunity in a mammal, wherein the method comprises administering to the mammal an effective amount of genetically modified cells encoding and expressing an anti-TIGIT antibody or a fragment thereof, wherein the anti-TIGIT antibody or the fragment thereof comprises an amino acid sequence selected from SEQ ID NO:7-18.

[0055] Exemplary implementations of this application include:

[0056] 1. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to the extracellular domain of TIGIT, and its binding affinity is determined to be better than 10 nM by SPR analysis, wherein...

[0057] (a) The HC contains

[0058] CDR1H, wherein CDR1H contains the amino acid sequence GYTFSRYWIE (SEQ ID NO:1),

[0059] CDR2H, wherein CDR2H contains the amino acid sequence EIFPGSGGTNYNEKFKG (SEQ ID NO:2), and

[0060] CDR3H, wherein CDR3H contains the amino acid sequence HLGALDY (SEQ ID NO:3);

[0061] (b) The LC includes

[0062] CDR1L, wherein CDR1L contains the amino acid sequence SASSVSYIH (SEQ ID NO:4),

[0063] CDR2L, wherein CDR2L contains the amino acid sequence RTSNLAS (SEQ ID NO:5), and

[0064] CDR3L, which contains the amino acid sequence QQYHSNPWT (SEQ ID NO:6).

[0065] 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0066] 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, further comprising a human receptor framework.

[0067] 4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the HC variable region sequence comprises an amino acid sequence selected from: SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from: SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0068] 5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the LC variable region sequence comprises an amino acid sequence selected from: SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from: SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0069] 6. The antibody or its antigen-binding fragment according to claim 5, wherein...

[0070] 1) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:11;

[0071] 2) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:8; and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:12;

[0072] 3) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:7; and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:12; or

[0073] 4) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:9; and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:10.

[0074] 7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7 and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12.

[0075] 8. The antibody or antigen-binding fragment thereof according to any one of items 1-7, wherein the antibody is an IgG isotype.

[0076] 9. An antibody or an antigen-binding fragment thereof according to any one of claims 1-8, wherein the antigen-binding fragment comprises any one of the following: Fab, F(ab')2, Fab', scFv, Fv, Fd, dAb and diabody.

[0077] 10. A bispecific antibody comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 and a second antibody or antigen-binding fragment thereof.

[0078] 11. The bispecific antibody according to claim 10, wherein the second antibody or its antigen-binding fragment specifically binds to a tumor antigen expressed on the surface of tumor cells or an immune checkpoint protein expressed on the surface of immune cells or tumor cells, wherein the tumor antigen or the immune checkpoint protein comprises any one of the following: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD 28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; Human Papillomavirus-E6; Human Papillomavirus-E7; JAM-3; KID3; KID31; KSA(17-1A) ); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosyltransferase; tumor suppressor M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; VEGFR; 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; galactolectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; and VSIG8.

[0079] 12. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of items 1-9, wherein a therapeutic agent is attached.

[0080] 13. The conjugate according to claim 12, wherein the therapeutic agent is a cytotoxin or a radioisotope.

[0081] 14. A composition comprising an antibody or an antigen-binding fragment thereof as described in any one of items 1-9, a bispecific antibody as described in item 10 or 11, or a conjugate as described in item 12 or 13, and a pharmaceutically acceptable excipient.

[0082] 15. Lymphocytes derived from the subject and subjected to in vitro treatment with an antibody or antigen-binding fragment thereof as described in any one of items 1-9.

[0083] 16. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in any one of items 1-9.

[0084] 17. An expression vector comprising the nucleic acid as described in item 16.

[0085] 18. Use of the antibody or antigen-binding fragment thereof as described in any one of items 1-9, the bispecific antibody as described in item 10 or 11, the composition as described in item 14, or the lymphocyte as described in item 15 in the preparation of a medicament for treating cancer in a subject.

[0086] 19. The use according to item 18, wherein the cancer is selected from one or more of the following: lymphoma, melanoma, colorectal adenocarcinoma, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, stomach cancer, and clear cell renal cell carcinoma.

[0087] 20. The use according to item 18 or 19, wherein the antibody or its antigen-binding fragment, bispecific antibody, polypeptide, conjugate, composition or lymphocyte is administered in combination with one or more antibodies or antibody fragments or anticancer agents, said antibody or antibody fragment or anticancer agent being selected from any one or more of the following: antibodies against checkpoint molecules or their receptors, anti-epidermal growth factor receptor (EGFR) agents, EGFR tyrosine kinase (TK) inhibitors, alkylating agents and topoisomerase inhibitors. Attached Figure Description

[0088] Figure 1 SDS-PAGE analysis results of purified antibody.

[0089] Figure 2 ELISA results to determine the binding affinity of the antibody to human TIGIT / MIgG2aFc protein.

[0090] Figure 3 Flow cytometry analysis of the binding affinity of antibodies to Jurkat cells expressing human TIGIT.

[0091] Figure 4 The study showed a high binding affinity between VH2+VL4 and macaque TIGIT.

[0092] Figures 5A-5B The interaction between TIGIT and CD155 on CD4+ cells (4A) and CD8+ cells (4B) was effectively blocked by humanized anti-TIGIT antibody.

[0093] Figure 6The study demonstrated that the anti-TIGIT antibody VH2+VL4 and tiragolumab monoclonal antibody inhibited tumor growth in vivo. Detailed Implementation

[0094] This disclosure provides antibodies and fragments thereof that bind to TIGIT proteins, particularly human TIGIT proteins or peptides. This disclosure also relates to the use of said antibodies and fragments thereof for enhancing the activation of the immune system against, for example, cancer cells.

[0095] This disclosure further provides a method for manufacturing anti-TIGIT antibodies, a polynucleotide encoding anti-TIGIT antibodies, and a cell containing the polynucleotide encoding anti-TIGIT antibodies.

[0096] 1. Definition

[0097] It should be understood that this disclosure is not limited to the aspects described herein, and may of course vary. It should also be understood that, as used herein, terminology is used only to describe a particular aspect and is not intended to be limiting, as the scope of this disclosure is limited only by the appended claims.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the techniques to which they pertain. All technical and patent disclosures referenced herein are incorporated herein by reference in their entirety. Unless otherwise stated, those skilled in the art will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the scope of the art. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Harlow and Lane, eds. (1999) Antibodies, A Laboratory Manual. MONCLONAL ANTIBODIES: A PRACTICAL APPROACH (Shepherd, P. et al., eds., 2000) Oxford University Press, USA, New York, NY.

[0099] The term "TIGIT" is an abbreviation for T-cell immune receptor with Ig and ITIM domains, also known as WUCAM, Vstm3, and VSIG9. It consists of an extracellular immunoglobulin variable group (IgV) domain, a type I transmembrane domain, and an intracellular domain possessing a typical immunoreceptor tyrosine inhibitory motif (ITIM) and immunoglobulin tyrosine tail (ITT) motif. TIGIT is a member of the poliovirus receptor / nectin family, a subset of the immunoglobulin superfamily. TIGIT is an immunoreceptor inhibitory checkpoint involved in tumor immune surveillance. TIGIT competes with the immune activator receptor CD226 (DNAM-1) for the same set of ligands: CD155 (PVR or poliovirus receptor) and CD112 (nectin-2 or PVRL2). However, TIGIT has a much weaker affinity for PVRL2 and PVRL3 than for PVR.

[0100] "PVR" is short for poliovirus receptor, also known as CD155, Necl5, and Tage4. PVR is a cell surface adhesion molecule that is sharply overexpressed in several human malignancies, while its expression is low or absent in most healthy tissues. Consistent with the biological characteristics of PVR, its overexpression promotes tumor cell invasion, migration, and proliferation, and is associated with poor prognosis and enhanced tumor progression.

[0101] As used herein, the term "anti-TIGIT antibody" refers to an antibody capable of specifically binding to TIGIT (such as human TIGIT). The advantage is that the specific binding of anti-TIGIT antibodies to TIGIT provides sufficient affinity for diagnosis and / or treatment. Preferably, the anti-TIGIT antibody competes with PVR and / or other TIGIT ligands for binding to TIGIT.

[0102] As used in this disclosure, the term "antibody," also known as "immunoglobulin," encompasses antibodies that have the structural features of natural antibodies and antibody-like molecules that have structural features different from those of natural antibodies but exhibit binding specificity to TIGIT molecules. The term antibody is intended to encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0103] The terms “heavy chain” (“HC”), “light chain” (“LC”), “light chain variable region” (“VL”), “heavy chain variable region” (“VH”), and “framework region” (“FR”) refer to the domains in naturally occurring immunoglobulins and the corresponding domains in synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is a tetramer with two light chains and two heavy chains. The amino-terminal (“N”) portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl terminus (“C” portion) of each chain is defined as the constant region. The light chain has a single constant domain, while the heavy chain typically has three constant domains and a hinge region. Therefore, the structure of the light chain of the naturally occurring IgG molecule is N-VL-CL-C, and the structure of the IgG heavy chain is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region). The variable region of the IgG molecule consists of the complementarity-determining region (CDR) (containing residues that contact the antigen) and non-CDR segments (called framework segments, which maintain the structure and determine the position of the CDR loop). Therefore, the VL and VH domains have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.

[0104] In natural antibodies, variability is unevenly distributed across the variable regions. It is concentrated in three segments of the light and heavy chain variable regions, known as complementarity-determining regions (CDRs) or hypervariable regions. A CDR on the heavy chain can be called CDRnH, where "n" is an integer and does not indicate the order of the CDRs on the heavy chain. Similarly, a CDR on the light chain can be called CDRnL, where "n" is an integer representing the CDR and does not indicate the order of the CDRs on the light chain. More highly conserved portions of the variable regions are called frames (FRs). The variable regions of both the natural heavy and light chains each contain four FR regions linked by three CDRs. The CDRs in each chain are tightly linked by FR regions and, together with CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site [see Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)]. Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody-dependent cytotoxicity (ADCC).

[0105] As used herein, the term "antigen-binding fragment" (or simply "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a TIGIT molecule, such as human TIGIT). The antibody fragment comprises only a portion of the complete antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions that would normally be associated with the portion when it is present in the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabody antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0106] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site and a residual Fc fragment, the name reflecting their ease of crystallization. The "Fab" fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The "Fab'" fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. "Fab'-SH" refers to Fab' where the cysteine ​​residues of the constant domain have free thiol groups. The "F(ab')" fragment is produced by the cleavage of the hinge cysteine ​​disulfide bond in the pepsin digestion product "F(ab')2".

[0107] The “Fd” fragment consists of VH and CH1 domains. The “dAb” fragment (Ward et al., (1989) Nature 341:544-546) consists of VH domains. Separate complementarity-determining regions (CDRs) and combinations of two or more separate CDRs can optionally be joined by synthetic linkers.

[0108] The “Fv” fragment consists of the VL and VH domains of the antibody single arm. The single-chain Fv (scFv) consists of a heavy chain variable region and a light chain variable region, which are covalently linked into a single-chain polypeptide chain by a flexible peptide linker.

[0109] The term "bimeric antibody" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment contains a heavy chain variable region (VH) linked to a light chain variable region (VL) within the same polypeptide chain (VH-VL). By using a linker (which is too short to allow pairing between two domains on the same chain), the domain is forced to pair with a complementary domain of the other chain, creating two antigen-binding sites. Bimeric antibodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-48 (1993).

[0110] These antibody fragments are obtained using conventional techniques known to those skilled in the art, such as recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0111] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or mutations that occur during the preparation of monoclonal antibodies, such variants are usually present in small amounts).

[0112] As used herein, the term "chimeric antibody" means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., the mouse Fc constant region) is replaced by the Fc constant region of an antibody from another species (e.g., the human Fc constant region) using recombinant DNA technology. See, for example, Robinson et al., PCT / US86 / 02269; Morrison et al., European Patent Application 173,494.

[0113] As used herein, the term "humanized antibody" refers to an antibody comprising a human framework region and one or more core receptors (CDRs) derived from a non-human (e.g., mouse, rat, rabbit, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is called the "donor," while the human immunoglobulin providing the framework is called the "acceptor." In one respect, all CDRs are derived from the donor immunoglobulin within the humanized immunoglobulin. Thus, apart from the possible CDRs, all portions of the humanized immunoglobulin are substantially identical to the corresponding portions of the natural human immunoglobulin sequence. Humanized antibodies can be constructed using genetic engineering techniques (see, for example, U.S. Patent No. 5,585,089).

[0114] "Acceptor human framework" refers to a framework containing the amino acid sequence of a light chain variable region (VL) framework or a heavy chain variable region (VH) framework derived from the human immunoglobulin framework or the human common framework. Receptor human frameworks "derived from" the human immunoglobulin framework or the human common framework may contain the same amino acid sequence, or they may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 1-10, 2-9, 3-8, 4-7, or 5-6.

[0115] "Human common framework" is a framework representing the most common amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from an isotype of the variable region sequence. Generally, the isotype of said sequence is an isotype as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In some embodiments, for VL, said isotype κI as described in Kabat et al. (ibid.). In some embodiments, for VH, said isotype III as described in Kabat et al. (ibid.).

[0116] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this technology may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific in vitro mutagenesis or by in vivo somatic mutations). However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from a germline of another mammalian species (such as a rabbit) has been transplanted into a human frame sequence. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially every part of the protein (e.g., CDR, frame, CL, CH domains (e.g., CH1, CH2, CH3), hinge, VL, VH) is substantially non-immunogenic in humans, with only minor sequence variations or mutations. Therefore, human antibodies differ from chimeric or humanized antibodies. It should be noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes.

[0117] As used herein, the phrase "bispecific antibody," "bispecific antigen-binding antibody," or "bifunctional antibody" refers to an artificial hybrid antibody having two distinct heavy / light chain pairs and two distinct binding sites. For the purposes of this disclosure, a "bispecific antibody" specifically binds to TIGIT and another antigen, such as a tumor antigen expressed on tumor cells.

[0118] "Conjugate" is an antibody conjugated with one or more heterologous molecules, including but not limited to cytotoxic agents.

[0119] "Blocking" antibodies or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigens they bind to. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0120] As used herein, the term "isolated" refers to molecular or biological or cellular material that is substantially free of other material. For example, nucleic acids or peptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally present as fragments and are not found in their native state. The term "isolated" is also used herein to refer to polypeptides isolated from other cellular proteins and is intended to encompass both purified and recombinant polypeptides.

[0121] As used herein, the percentage of “homology” or “identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences being identical or having a specified percentage of identical nucleotide or amino acid residues, for example, having at least 80% identity in a specified region (e.g., the nucleotide sequence encoding the antibody described herein or the amino acid sequence of the antibody described herein), preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity. Homology can be determined by comparing positions in the sequences, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or the number of homologous positions shared by the sequences. Alignment and determination of the percentage of homology or sequence identity can be performed using software programs known in the art. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. The preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0122] “Affinity” refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to inherent binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can be measured by conventional methods known in the art, including, for example, Biacore, radioimmunoassay (RIA), and ELISA.

[0123] The affinity of molecule X for its partner Y can usually be represented by the equilibrium dissociation constant (KD), which is expressed as a ratio k. off / k on (k d / k a) Calculation. See, for example, Chen, Y., et al., (1999) J. MoI Biol 293:865-881. Low-affinity antibodies typically bind antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind antigens more quickly and tend to maintain binding for a longer period. In one embodiment of this disclosure, “dissociation rate (k d The term "association rate" is measured using surface plasmon resonance determination. According to this disclosure, "association rate" or "association rate (kJ / kM)" is used. a ) or "k on "It can also be determined using the same surface plasmon resonance technique and calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software) by simultaneously fitting association and dissociation sensor maps."

[0124] As used herein, the term “EC50” refers to the concentration of the antibody or its antigen-binding fragment that binds TIGIT and / or induces a response at 50% of maximum binding or response, i.e., at half the baseline, in an in vitro or in vivo assay.

[0125] The terms “cancer,” “neoplasm,” and “tumor” are used interchangeably in this disclosure to refer to a growth or tumor resulting from the abnormal, uncontrolled growth of cells that makes it pathogenic to the host organism. In some embodiments, cancer refers to a benign tumor that has been confined to a localized area. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In some embodiments, cancer is associated with a specific cancer antigen.

[0126] As used herein, “treating” or “treatment” for a subject’s disease refers to a method for obtaining a beneficial or desired outcome, including but not limited to one or more of the following: reduction or improvement of one or more symptoms, reduction of the extent of the condition (including disease), stabilization (i.e., non-deterioration) of the condition (including disease), delay or slowing of the condition (including disease), progression, improvement or mitigation of the condition (including disease), and status and remission (whether partial or complete), whether detectable or undetectable.

[0127] A "pharmaceutically acceptable carrier" is a carrier that forms a pharmaceutical formulation with the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0128] The term "packaging insert" is used to refer to the instructions for use typically included in the commercial packaging of therapeutic products. Generally, packaging inserts contain information about the use of therapeutic products, such as indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings.

[0129] This disclosure will be described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto, but is defined only by the claims. The term "comprising" as used in this specification and claims does not exclude other elements or steps. When referring to singular nouns, indefinite or definite articles are used, such as "a" or "an," "the," unless otherwise specified, which includes the plural form of the noun.

[0130] 2. Anti-TIGIT antibody and its preparation method

[0131] This disclosure covers isolated anti-TIGIT antibodies or fragments thereof, comprising a polynucleotide sequence encoding an anti-TIGIT antibody or fragment thereof.

[0132] Isolated anti-TIGIT antibodies or fragments thereof bind with high affinity to TIGIT molecules expressed on cells (e.g., cancer cells), promoting an effective immune response against cancer cells. The antibodies and immunomodulatory fragments thereof provided in this disclosure can enhance the activity of the immune system, thereby providing important therapeutic and diagnostic agents for pathological conditions associated with the expression and / or activity of TIGIT molecules. In one aspect, this disclosure provides an isolated antibody or antigen-binding fragment thereof comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence. The antibody binds to the extracellular domain of TIGIT with a binding affinity better than 10 nM or about 10 nM, better than 8 nM or about 8 nM, better than 6 nM or about 6 nM, better than 4 nM or about 4 nM, better than 2 nM or about 2 nM, better than 1 nM or about 1 nM, better than 0.8 nM or about 0.8 nM, better than 0.6 nM or about 0.6 nM, better than 0.4 nM or about 0.4 nM, better than 0.2 nM or about 0.2 nM, as determined by SPR analysis, for example, about 0.1-0.2 nM, about 0.1-0.18 nM, about 0.1-0.13 nM, about 0.1 nM, 0.11 nM, 0.12 nM or better, as determined by SPR analysis.

[0133] In some embodiments, this disclosure provides an antibody or an antigen-binding fragment thereof comprising at least one of the following:

[0134] (a) A CDR1H sequence containing GYTFSRYWIE (SEQ ID NO:1),

[0135] (b) A CDR2H sequence containing EIFPGSGGTNYNEKFKG (SEQ ID NO:2),

[0136] (c) A CDR3H sequence containing HLGALDY (SEQ ID NO:3),

[0137] (d) A CDR1L sequence containing SASSVSYIH (SEQ ID NO:4),

[0138] (e) A CDR2L sequence containing RTSNLAS (SEQ ID NO:5), and

[0139] (f) A CDR3L sequence containing QQYHSNPWT (SEQ ID NO:6).

[0140] In some embodiments, this application provides an antibody or an antigen-binding fragment thereof, wherein

[0141] (a) The HC contains

[0142] The CDR1H sequence containing GYTFSRYWIE (SEQ ID NO:1)

[0143] The CDR2H sequence containing EIFPGSGGTNYNEKFKG (SEQ ID NO:2), and

[0144] The CDR3H sequence containing HLGALDY (SEQ ID NO:3).

[0145] (b) The LC includes

[0146] The CDR1L sequence containing SASSVSYIH (SEQ ID NO:4)

[0147] The CDR2L sequence containing RTSNLAS (SEQ ID NO:5), and

[0148] The CDR3L sequence containing QQYHSNPWT (SEQ ID NO:6).

[0149] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody or its antigen-binding fragment of the present disclosure further comprises a human receptor framework. In some embodiments, the human receptor framework is derived from a human immunoglobulin framework or a human common framework. In some embodiments, the human receptor framework comprises a VL isotype κI framework sequence and a VH isotype III framework sequence. Typically, the isotype sequences are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for VL, the isotype is the κI isotype described by Kabat et al. as described above. In some embodiments, for VH, the isotype is the III isotype described by Kabat et al. as described above.

[0150] In some embodiments, the antibody or its antigen-binding fragment comprises a human common framework. In some embodiments, the antibody or its antigen-binding fragment comprises a human common framework with amino acid sequence variations, such as 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acid variations.

[0151] In some embodiments, the antibody or antigen-binding fragment of this application comprises an HC variable region sequence containing the amino acid sequence shown in SEQ ID NO:7, 8, or 9, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7, 8, or 99. In some embodiments, the antibody or antigen-binding fragment of this application comprises an LC variable region sequence containing the amino acid sequence shown in SEQ ID NO:10, 11, or 12, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:10, 11, or 12. In some embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. In some embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:9, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:10.

[0152] In some embodiments, the antibody or antigen-binding fragment of this application comprises an HC sequence containing the amino acid sequence shown in SEQ ID NO:13, 14, or 15, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:13, 14, or 15. In some embodiments, the antibody or antigen-binding fragment of this application comprises an LC sequence containing the amino acid sequence shown in SEQ ID NO:16, 17, or 18, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:16, 17, or 18. In some embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO:14, and the LC sequence comprises the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO:13 and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO:15 and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:16.

[0153] In some embodiments, the antibody is an IgG isotype, such as IgG1, IgG2, or IgG4 isotypes. In some embodiments, the antigen-binding fragment is selected from any one or more of the following: Fab, F(ab')2, Fab', scFv, and Fv. In some embodiments, the antibody or antigen-binding fragment of this disclosure is a blocking antibody or antagonist antibody that inhibits or reduces the biological activity of the TIGIT molecule it binds to. Preferably, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the TIGIT molecule.

[0154] The anti-TIGIT antibody of this application is preferably monoclonal. Also covered within the scope of this disclosure are the Fab, Fab', Fab'-SH, and F(ab')2 fragments of the anti-TIGIT antibody provided herein. These antibody fragments can be produced by conventional methods such as enzymatic digestion, or by recombinant technology. The anti-TIGIT antibody and its fragments can be used for diagnostic and therapeutic purposes, including the diagnosis and treatment of cancer.

[0155] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, meaning that individual antibodies comprising the population are identical, except that a small amount of naturally occurring mutations may be present. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of different antibodies. The monoclonal anti-TIGIT antibody of this application can be prepared using hybridoma methods or recombinant DNA methods (US Patent No. 4,816,567).

[0156] In the hybridoma approach, mice or other suitable host animals, such as hamsters, are immunized with the whole TIGIT molecule or a portion thereof (e.g., a polypeptide containing the extracellular domain of TIGIT), along with an adjuvant. TIGIT molecules or polypeptides containing the extracellular domain of TIGIT molecules can be prepared using methods known in the art. In one embodiment, animals are immunized with a polypeptide containing the extracellular region (ECD) of TIGIT, said extracellular region fused to the Fc portion of an immunoglobulin heavy chain. In one embodiment, animals are immunized with a TIGIT-IgG1 fusion protein. Two weeks later, the animals are boosted. Seven to fourteen days later, blood is collected from the animals, and the serum anti-TIGIT titer is determined. Animals are boosted until the titer stabilizes. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0157] The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. Preferred myeloma cells are those that are efficiently fused, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to culture media (such as HAT medium). Preferred myeloma cell lines are murine myeloma cell lines, such as SP-2 or X63-Ag8-653 cells. (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The use of human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies is also described.

[0158] The production of monoclonal antibodies against TIGIT in the culture medium for hybridoma cells is measured. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0159] The binding affinity of the monoclonal antibody can then be determined using conventional methods in the art. Once it has been identified that hybridoma cells can produce antibodies with the desired specificity, affinity, and / or activity, they can be subcloned using a limited dilution procedure and cultured using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0160] Suitable culture media for the purposes described above include, for example, D-MEM or RPMI-1640 media. Additionally, hybridoma cells can be grown in animals as ascites tumors. Subclonal secreted monoclonal antibodies are appropriately isolated from the culture medium, ascites fluid, or serum using routine immunoglobulin purification procedures.

[0161] The anti-TIGIT antibody of this application can be manufactured by screening synthetic antibody clones with one or more desired activities using a combinatorial library. Generally, synthetic antibody clones are selected by screening a phage library containing phages displaying different fragments of the antibody variable region (Fv), the Fv fragments being fused to the phage coat protein. This phage library is then panned using affinity chromatography against the target antigen. The Fv fragment expressed by the clone binds to the target antigen, adsorbing onto it and thus separating it from unbound clones in the library. The bound clones are then eluted from the antigen and can be further enriched through additional cycles of antigen adsorption / elution. Any anti-TIGIT antibody disclosed herein can be obtained by designing a suitable antigen screening procedure, selecting a target phage clone, and then constructing a full-length anti-TIGIT antibody clone using the Fv sequence from the target phage clone and a suitable constant region (Fc) sequence as described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Edition, NIHP Publication 91-3242, Bethesda MD (1991), Volumes 1-3.

[0162] Repertoires of the VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then searched for antigen-binding clones, as described in Winter et al., Ann. Rev. Immunol, 12:433-455 (1994). Libraries from immune sources can provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned to provide single-source human antibodies to a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be synthesized by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode a highly variable CDR3 region, and performing rearrangements in vitro, as described in Hoogenboom and Winter, J. MoI Biol, 227:381-388 (1992).

[0163] Antibodies derived from immature libraries (natural or synthetic) can have moderate affinity, but affinity maturation can also be simulated in vitro by constructing secondary libraries and reselecting from them. For example, this can be achieved by randomly introducing mutations in vitro using a fault-prone polymerase (reported in Leung et al., Technique, 1:11-15 (1989)) in the methods of Hawkins et al., J. MoL Biol., 226:889-896 (1992) or Gram et al., Proc. Natl. Acad. Sci USA, 89:3576-3580 (1992). Alternatively, affinity maturation can be performed in selected single Fv clones by randomly mutating one or more CDRs (e.g., using PCR and primers carrying random sequences covering the target CDR), and screening for clones with higher affinity. Another effective approach is to recombine the selected VH or VL domains displayed by phages with a library of naturally occurring V domain variants obtained from unimmunized donors, and screen for higher affinity in several rounds of strand shuffling, as described in Marks et al., Biotechno L., 10:779-783 (1992).

[0164] For TIGIT, even with slight differences in affinity, selection can be made between phage antibodies with different affinities. However, random mutation of the selected antibody (e.g., as performed in some of the affinity maturation techniques described above) can produce many mutants, most of which bind to the antigen, with a few exhibiting higher affinity. To preserve all the higher-affinity mutants, the phage can be incubated with an excess of biotinylated TIGIT, but at a molar concentration below the target molar affinity constant for TIGIT. The high-affinity-binding phage can then be captured by streptavidin-coated paramagnetic beads. This “balanced capture” allows for antibody selection based on binding affinity, with a sensitivity that allows isolation of mutant clones with up to twice the higher affinity from a large excess of low-affinity phage.

[0165] Anti-TIGIT clones can be selected based on activity. In one embodiment, this disclosure provides an anti-TIGIT antibody that blocks the binding between the TIGIT receptor and its ligand. Anti-TIGIT antibodies of this disclosure having the properties described herein can be obtained by screening anti-TIGIT hybridoma clones for the desired properties using any convenient method. For example, if the desired antibody is an anti-TIGIT monoclonal antibody that blocks or does not block the binding of the TIGIT receptor to the TIGIT ligand, the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, wherein the wells are coated with TIGIT, a solution of antibody with an excess of the TIGIT receptor is plated on the coated plate, and the bound antibody is detected by an enzymatic reaction, for example, by contacting the bound antibody with an HRP-conjugated anti-Ig antibody or a biotinylated anti-Ig antibody and performing an HRP colorimetric reaction (e.g., by colorimetrically developing the plate with streptavidin-HRP and / or hydrogen peroxide, and detecting the HRP colorimetric reaction spectrophotometrically at 490 nm using an ELISA reader).

[0166] 3. Isolation of polynucleotides, vectors, host cells, and recombinant methods

[0167] This disclosure provides isolated polynucleotides, vectors, or host cells comprising the coding sequences of the anti-TIGIT antibody or fragments thereof disclosed herein. In some embodiments, the anti-TIGIT antibody is a hybridoma-derived monoclonal antibody or a phage-displayed Fv clone of this disclosure. In some embodiments, the DNA encoding the hybridoma-derived monoclonal antibody or phage-displayed Fv clone of this disclosure is isolated and sequenced using routine procedures (e.g., by using oligonucleotide primers designed to specifically amplify the target heavy and light chain coding regions from a hybridoma or phage DNA template). Once isolated, the DNA can be placed in an expression vector and then transfected into host cells (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce immunoglobulins) to obtain the desired monoclonal antibody synthesis in the recombinant host cells.

[0168] The DNA encoding the Fv clone disclosed herein can be combined with a known DNA sequence encoding a heavy chain and / or light chain constant region (e.g., a suitable DNA sequence may be obtained from Kabat et al. (ibid.)) to form a clone encoding a full-length or partial-length heavy chain and / or light chain. It should be understood that any isotype constant region may be used for the purposes described, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions may be obtained from any human or animal species. An Fv clone, derived from variable region DNA of one animal species (such as human), is then fused with constant region DNA of another animal species to form a “hybrid,” as defined herein in the terms “chimeric” and “hybrid” antibodies, which include coding sequences for a full-length heavy chain and / or light chain. In a preferred embodiment, an Fv clone derived from human variable DNA is fused with human constant region DNA to form coding sequences for a full-human, full-length, or partial-length heavy chain and / or light chain.

[0169] The DNA encoding anti-TIGIT antibodies derived from hybridomas of this disclosure can also be modified, for example, by replacing homologous mouse sequences derived from hybridoma clones with coding sequences for human heavy and light chain constant domains (e.g., the method in Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851-6855 (1984)). The DNA encoding antibodies or fragments derived from hybridoma or Fv clones can be further modified by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. In this way, “chimeric” or “hybrid” antibodies having binding specificity to antibodies derived from Fv clones or hybridoma clones of this disclosure are prepared.

[0170] To recombinantly generate the antibodies of this disclosure, the nucleic acid encoding it is isolated and inserted into a reproducible vector for further cloning (DNA amplification) or for expression. The DNA encoding the antibody is readily isolated and sequenced using standard procedures, e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody. Many types of vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of prokaryotic or eukaryotic (typically mammalian) origin. It should be understood that any isotype constant region can be used for the purposes described, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species.

[0171] 4. Conjugates and their preparation methods

[0172] The anti-TIGIT antibody or fragment thereof disclosed herein, together with one or more other molecules (such as toxins, such as calicheamicin, maytansinoids, dolastatins, aurostatins, trichothecene, and CC1065, and derivatives of said toxins having toxic activity), radioisotopes, and immunomodulators, are considered in this application.

[0173] In some embodiments, the conjugate is used to treat T-cell lymphoma, and the conjugate comprises an antibody (full-length or fragment) of the present disclosure conjugated with one or more maytansine alkaloid molecules. Maytansine alkaloids are mitotic inhibitors that act by inhibiting microtubule polymerization. Maytansine was originally isolated from the East African shrub *Maytenus serrate* (US Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansine alkaloids, such as maytanol and C-3 maytanol ester (US Patent No. 4,151,042). Immunohistochemical conjugates containing maytansine alkaloids, methods of their preparation, and their therapeutic uses are disclosed, for example, in US Patents 5,208,020 and 5,416,064 and European Patent EP 0 425 235 B1 (the disclosures of which are expressly incorporated herein by reference). The antibody-matenine alkaloid conjugate can be manufactured using a variety of bifunctional protein conjugates, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), iminothiacyclopentane (IT), and bifunctional derivatives of imine esters. In some embodiments, the immunoconjugate comprises an antibody of the present disclosure conjugated to a saurus toxin or a saurus toxin peptide analog and derivative, auristatin (US Patent Nos. 5,635,483; 5,780,588). To selectively destroy tumors, the antibody may contain highly radioactive atoms. A variety of radioisotopes can be used to generate radioconjugated antibodies. Radioactive or other labeling can be incorporated into the conjugate in known ways. For example, the peptide can be biosynthesized or synthesized using suitable amino acid precursors via chemical amino acid synthesis, including, for example, replacing hydrogen with fluorine-9. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0174] In some embodiments, the conjugate is used to treat T-cell lymphoma and comprises an antibody (full-length or fragment) of the present disclosure conjugated with one or more immunomodulatory agents, wherein the immunomodulatory agents may work synergistically with the antibody (full-length or fragment) to enhance the immune response against antigens and abnormal cells, including tumor cells. In some embodiments, the immunomodulator is selected from any of the following: checkpoint inhibitors (such as atezolizumab, avelumab, cimiplimab, durvalumab, ipilimumab, nivolumab, pembrolizumab), cytokines (such as aldesleukin, granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2B, Pre-IFNα-2B), agonists and adjuvants (such as imiquimod or polyICLC), or molecules with the same function.

[0175] Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid-phase synthesis methods well-known in the field of peptide chemistry. The auristatin drug moieties can be prepared according to the following methods: US 5635483; US 5780588. See also Doronina (2003) Nat Biotechnol 21(7):778-784.

[0176] This disclosure further considers the formation of immunoconjugates between antibodies and compounds with nucleolytic activity (e.g., ribonucleases or DNA endonucleases such as deoxyribonuclease; DNase).

[0177] 5. Antibody fragments and their preparation methods

[0178] This disclosure includes antibody fragments. The antibody fragments are immunologically active fragments of the anti-TIGIT antibody of this disclosure. In some cases, there are advantages to using antibody fragments instead of the whole antibody. The smaller size of the fragment allows for rapid clearance and makes it easier for it to enter solid tumors.

[0179] Various techniques have been developed for generating antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be generated directly from recombinant host cells. Fab, Fv, and ScFv antibody fragments can be expressed and secreted in and from *E. coli*, thus allowing for convenient mass production. Antibody fragments can be isolated from the aforementioned antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from *E. coli* and chemically conjugated to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. U.S. Patent 5,869,046 describes Fab and F(ab')2 fragments with increased in vivo half-life, which contain rescue receptor-binding epitope residues. Other techniques for generating antibody fragments will be apparent to those skilled in the art.

[0180] In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent 5,571,894; and 5,587,458. Fv and scFv are the only species with an intact binding site and no constant region; therefore, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusion of effector proteins at the amino or carboxyl termini of the scFv. See Antibody Engineering, ed. Borrebaeck, ibid. The antibody fragment can also be a “linear antibody,” for example, as described in U.S. Patent 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0181] 6. Humanized antibodies and human antibodies

[0182] The anti-TIGIT antibody disclosed herein is, in some embodiments, a humanized antibody. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therefrom from a non-human source. These non-human amino acid residues are generally referred to as “introduced” residues, which are typically derived from the “introduced” variable region. Humanization can be essentially performed according to the methods of Winter and colleagues (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536) by replacing the corresponding sequence of the human antibody with a hypervariable region sequence. Thus, such a “humanized” antibody is a chimeric antibody (US Patent 4,816,567), in which a portion substantially smaller than the complete human variable region is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable residues and possibly some FR residues are replaced by similar site residues from rodent antibodies. The selection of the human variable regions (light and heavy chains) used to manufacture humanized antibodies is crucial for reducing antigenicity. According to the so-called “best-fit” approach, variable region sequences of rodent antibodies are screened against a whole library of known human variable region sequences. The human sequence closest to the rodent is then used as the human frame for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. MoI. Biol. 196:901). Another approach uses a specific frame derived from a common sequence of a specific subtype of a fully human antibody in either the light or heavy chain.

[0183] Further important is the humanization of the antibody while retaining its high affinity for the antigen and other advantageous biological properties. To achieve this goal, humanized antibodies are prepared according to a method involving the analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are universally available and familiar to those skilled in the art. Computer programs illustrating and displaying the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examining these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the acceptor and input sequences and bound to obtain desired antibody properties, such as increased affinity for TIGIT.

[0184] Transgenic animals (e.g., mice) in the absence of endogenous immunoglobulin production can still produce a complete library of human antibodies after immunization. For example, it has been described that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice completely suppresses the production of endogenous antibodies. Introducing human germline immunoglobulin gene arrays into such germline mutant mice will produce human antibodies upon antigen stimulation. See, for example, Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol, 7:33 (1993).

[0185] Gene shuffling can also be used to obtain human antibodies from non-human (e.g., rodent) antibodies, wherein the human antibodies have similar affinity and specificity to the starting non-human antibody. According to the method (also known as “epitope imprinting”), the heavy or light chain variable region of a non-human antibody fragment obtained via the phage display technology described above is replaced with a library of human V domain genes, creating a population of non-human chain / human chain scFv or Fab chimeras. Selection with antigens isolates the non-human chain / human chain chimera scFv or Fab, where the human chain restores the antigen-binding site disrupted when the corresponding non-human chain in the initial phage display clone was removed; that is, the epitope controls (imprints) the selection of the human chain chaperone. When the process is repeated to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93 / 06213, published April 1, 1993). Unlike the humanization of conventional non-human antibodies via CDR transplantation, this technique provides fully human antibodies without non-human FR or CDR residues.

[0186] 7. Bispecific antibodies and their preparation methods

[0187] A bispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigens, preferably human or humanized antibodies. In this disclosure, one binding specificity is against TIGIT, and the other is against any other antigen. Exemplary bispecific antibodies can bind to two different epitopes of the TIGIT protein. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing TIGIT, in which case the antibody has a TIGIT-binding arm and a cytotoxic agent-binding arm.

[0188] In some embodiments, the bispecific antibody has a TIGIT binding arm comprising the anti-TIGIT antibody of this disclosure or a fragment thereof, and an arm for binding to a tumor antigen or immune checkpoint protein. In some embodiments, the tumor antigen is selected from any one or more of the following: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; human Human papillomavirus-E6; Human papillomavirus-E7; JAM-3; KID3; KID31; KSA(17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosamine transferase; tumor suppressor M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor and VEGF receptor. In some embodiments, the immune checkpoint protein is selected from any one or more of the following: 2B4; 4-1BB; 4-1BB ligand; B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; galactoglobulin 9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; VSIG8.

[0189] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody). Methods for manufacturing bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities. Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (tetragenic hybridomas) have the potential to produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, typically accomplished by affinity chromatography, is cumbersome and yields low results. Depending on different and more preferred methods, an antibody variable region with the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence. The fusion is preferably fused to an immunoglobulin heavy-chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. Preferably, at least one fusion contains a first heavy-chain constant region (CH1) containing the site necessary for light chain binding. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the ratio of the three polypeptide fragments in the implementation scheme when using three polypeptide chains in unequal proportions in the construct provides optimal yield. However, when expressing at least two polypeptide chains in equal proportions results in high yield, or when the proportions are not particularly significant, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector.

[0190] In a preferred embodiment of the method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It has been found that the asymmetric structure facilitates the separation of the desired bispecific compound from unwanted combinations of immunoglobulin chains, as the presence of the immunoglobulin light chain in only half of the bispecific antibody provides a convenient separation method. The method is disclosed in WO 94 / 04690. Further details on the generation of the bispecific antibody can be found, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0191] 8. Pharmaceutical Composition

[0192] Therapeutic agents comprising the anti-TIGIT antibody fragments, polynucleotides, vectors, host cells, conjugates, or bispecific antibodies of the present disclosure are prepared for storage by means of the anti-TIGIT antibody, fragment, polynucleotide, vector, host cell, conjugate, or bispecific antibody of the present disclosure having the desired purity, along with optional physiologically acceptable vectors, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy 20th edition (2000)), in the form of an aqueous solution, lyophilized or otherwise desiccated. Acceptable carriers, excipients, or stabilizers are non-toxic to subjects at the doses and concentrations used and include buffers such as phosphates, citrates, histidine, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming anti-charge ions such as sodium; metal complexes; and / or nonionic surfactants such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0193] As required for the specific indications being treated, the formulations described herein may also contain more than one active compound, preferably those compounds that have complementary activities without adversely affecting each other. Such molecules are suitable to be present in combination in amounts effective for the intended purpose.

[0194] In colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions, the active ingredient may also be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively.

[0195] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the immunoglobulins of this disclosure, said matrix being in the form of a molded article, such as a film or microcapsule.

[0196] 9. Diagnostic and therapeutic uses of anti-TIGIT antibodies

[0197] On one hand, based on the specific binding of the antibodies disclosed herein to TIGIT, the antibodies disclosed herein can be used to detect and quantify TIGIT peptides in physiological samples, such as urine, plasma, cell lysates, and biopsy samples. Therefore, the anti-TIGIT antibodies disclosed herein can be used for diagnostic monitoring of TIGIT levels in tissues, for example, to determine cancer progression and / or the efficacy of a given treatment regimen. Those skilled in the art will understand that the TIGIT antibodies disclosed herein can be conjugated to detectable materials to facilitate detection. In some embodiments, the anti-TIGIT antibodies disclosed herein, or fragments thereof, are bound to a solid support to facilitate detection.

[0198] On the other hand, based on the specific binding of the antibodies disclosed herein to TIGIT, the antibodies disclosed herein can be used, for example, to separate, analyze or sort cells by affinity chromatography or immunoprecipitation, and to detect TIGIT peptides in fixed tissue samples or cell smear samples by immunohistochemistry, cytological analysis, ELISA or immunoprecipitation.

[0199] In some embodiments, the TIGIT molecule to be detected, quantified, or analyzed is human TIGIT protein or a fragment thereof. In some embodiments, the TIGIT protein or a fragment thereof is placed in a solution, such as a lysis solution or a solution containing subcellular fractions of broken cells, or is present on the surface of TIGIT-positive cells, or in a complex containing TIGIT and other cellular components.

[0200] The detection method disclosed herein can be used to detect the expression level of TIGIT peptides in biological samples both in vitro and in vivo. In vitro techniques for detecting TIGIT peptides include enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, immunoprecipitation, radioimmunoassay, and immunofluorescence (e.g., IHC). Furthermore, in vivo techniques for detecting TIGIT peptides include introducing a labeled anti-TIGIT antibody into a subject. By way of example only, the antibody can be labeled with a radiolabel, the presence and location of which in the subject can be detected by standard imaging techniques.

[0201] Other antibody-based methods for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels (such as glucose oxidase) and radioisotopes or other radioactive reagents, as well as fluorescent labels (such as fluorescein and rhodamine), and biotin.

[0202] The TIGIT antibody or fragment thereof disclosed herein can be used as a diagnostic reagent for any type of biological sample. In one aspect, the TIGIT antibody disclosed herein can be used as a diagnostic reagent for human biological samples. The TIGIT antibody can be used to detect TIGIT peptides in various standard assays. Such assays include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, flow cytometry, IHC, and immunometric assays.

[0203] This disclosure also provides for the prognostic (or predictive) use of anti-TIGIT antibodies and fragments thereof to determine whether a subject is at risk of having a medical disease or condition associated with increased TIGIT peptide expression or activity (e.g., detection of precancerous cells). Therefore, the anti-TIGIT antibodies and fragments thereof disclosed herein can be used for prognostic or predictive purposes to preventively treat an individual prior to the onset of a medical disease or condition (e.g., cancer) characterized by, or associated with, increased TIGIT peptide expression or activity.

[0204] Another aspect of this disclosure provides a method for determining TIGIT expression in a subject to screen for therapeutic or preventative compounds for a medical disease or condition (e.g., cancer) characterized by, or associated with, an increase in TIGIT peptide expression or activity.

[0205] In some embodiments, the aforementioned medical disease or condition is a precancerous condition or cancer, characterized by the expression or activity of the TIGIT peptide, or an increase in the expression or activity of the TIGIT peptide, or an association with an increase in the expression or activity of the TIGIT peptide. In some embodiments, prognostic assays can be used to identify subjects who have cancer or are at risk of developing cancer. Therefore, this disclosure provides a method for identifying diseases or conditions (e.g., cancer) associated with increased TIGIT peptide expression levels, wherein a test sample is obtained from a subject and the TIGIT peptide is detectable, wherein the presence of an increased TIGIT peptide level compared to a control sample predicts that the subject has a disease or condition (e.g., cancer) associated with the increased TIGIT peptide expression level or is at risk of developing said disease or condition (e.g., cancer).

[0206] On the other hand, this disclosure provides a method for determining whether a subject can be effectively treated with a therapeutic agent targeting a condition or ailment (e.g., cancer) associated with increased expression of the TIGIT peptide, wherein a biological sample is obtained from the subject and the TIGIT peptide is detected using a TIGIT antibody. The expression level of the TIGIT peptide in the biological sample obtained from the subject is determined and compared to the TIGIT expression level found in a biological sample obtained from a disease-free subject. Elevated TIGIT peptide levels in samples obtained from subjects suspected of having a disease or ailment, compared to samples obtained from healthy subjects, indicate a TIGIT-related disease or ailment (e.g., cancer) in the subject being tested.

[0207] In one aspect, this disclosure provides a method for monitoring the therapeutic efficacy of a drug on TIGIT peptide expression. Such an assay can be applied to drug screening and clinical trials. For example, the effectiveness of a drug in reducing TIGIT peptide levels can be monitored in clinical trials in subjects exhibiting elevated TIGIT expression, such as patients diagnosed with cancer. Drugs affecting TIGIT peptide expression can be identified by administering the drug and observing the response. In this way, the expression pattern of the TIGIT peptide can be used as a biomarker indicating the physiological response of a subject to the drug.

[0208] The foregoing are merely exemplary assays using the anti-TIGIT antibodies and fragments thereof disclosed herein. Other methods for the assay of TIGIT using antibodies or fragments thereof, now or in the future, are also included within the scope of this disclosure.

[0209] In one aspect, this disclosure provides a method for treating cancer, the method comprising administering to a subject requiring such treatment an effective amount of an anti-TIGIT antibody or a fragment thereof that specifically binds to TIGIT. The antibodies of this disclosure can be used to treat, inhibit, or delay the progression of a disease, symptom, or condition associated with, or with increased expression and / or activity of one or more antigen molecules including a TIGIT molecule, such as molecule TIGIT, ...

[0210] For the therapeutic use of the anti-TIGIT antibody or fragment thereof disclosed herein, the appropriate dose of the antibody (when used alone or in combination with other agents) will depend on the type of disease to be treated, the type of antibody, the severity and duration of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for single or multiple administrations to the patient. Depending on the type and severity of the disease, an antibody dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) is an appropriate dose to be administered to the patient, whether, for example, by single or multiple administrations alone, or by continuous infusion.

[0211] The antibodies disclosed herein can be used alone or in combination with other compositions for treatment. For example, the antibodies disclosed herein can be co-administered with another antibody, steroids (such as inhaled, systemic, or transdermal steroids), chemotherapeutic agents (including mixtures of chemotherapeutic agents), other cytotoxic agents, anti-angiogenic agents, cytokines, and / or growth inhibitors. Such combination therapies include co-administration (where two or more agents are included in the same or separate formulations) and separate administration, in which case the anti-TIGIT antibody or a fragment thereof disclosed herein may be administered before, during, and / or after administration of one or more other agents. The effective amount of co-administered therapeutic agents depends on factors such as the type of therapeutic agent to be used and the specific patient to be treated, and is usually determined by a physician or veterinarian.

[0212] 10. Reagent kits and products

[0213] This disclosure provides a diagnostic method for determining the expression level of TIGIT. In one specific aspect, this disclosure provides a kit for determining the expression level of TIGIT or the presence and / or amount of TIGIT. The kit comprises the anti-TIGIT antibody or a fragment thereof disclosed herein, along with instructions on how to use the kit, such as instructions for collecting samples and / or performing detection and / or analyzing the results. The kit can be used to detect the presence of TIGIT peptides in biological samples, such as bodily fluids, including but not limited to, sputum, serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, or blood, including biopsy samples of human tissue. The test sample can also be tumor cells, normal cells adjacent to a tumor, normal cells corresponding to a tumor tissue type, blood cells, peripheral blood lymphocytes, or combinations thereof.

[0214] In some embodiments, the kit may further comprise one or more other TIGIT antibodies besides the anti-TIGIT antibodies of this disclosure, which are capable of binding to TIGIT peptides in biological samples. The one or more TIGIT antibodies may be labeled. In some embodiments, the kit comprises, for example, a first antibody attached to a solid support that binds to the TIGIT peptide; and optionally 2) a second, different antibody that binds to the TIGIT peptide or the first antibody and is conjugated with a detectable label.

[0215] The kit may also contain, for example, buffers, preservatives, or protein stabilizers. The kit may also contain components necessary for detecting the detectable label, such as enzymes or substrates. The kit may also contain a control sample or a series of control samples that can be assayed and compared with the test sample. Each component of the kit may be contained in a separate container, and all multiple containers may be housed in a single package, with an insert on the package containing instructions on how to use the kit, such as instructions for collecting samples and / or performing detection and / or analyzing results.

[0216] In another aspect, this disclosure provides an article comprising materials for treating, preventing, and / or diagnosing the aforementioned conditions. The article comprises a container and a label or packaging insert on or attached to the container, the label or packaging insert having written instructions such as therapeutic indications, administration methods, and warnings. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. The container contains a composition comprising an anti-TIGIT antibody or fragment thereof of this disclosure, the composition, alone or in combination with another composition, being effective for treating, preventing, and / or diagnosing a medical disease or condition (e.g., cancer) characterized by increased expression and / or activity of one or more molecules comprising a TIGIT polypeptide, or associated with increased expression and / or activity of one or more molecules.

[0217] The article may comprise: (a) a first container containing a composition comprising the antibody of the present disclosure; and (b) a second, third, or fourth container having a composition comprising another active ingredient. Additionally, the article may further comprise a container containing pharmaceutically acceptable buffers, such as water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0218] 11. Treatment methods

[0219] The anti-TIGIT antibody or fragment thereof disclosed herein may be used for specific treatment methods. This disclosure further includes antibody-based therapies involving the administration of an effective amount of the antibody of this disclosure or its antigen-binding fragment, bispecific antibody, peptide, conjugate, composition, article, or kit to a patient, such as a human patient or non-human primate, to treat one or more of the diseases or conditions described herein.

[0220] In some embodiments, the patient is a patient with a tumor. In some embodiments, the patient has an infection. In one embodiment, the patient has tumor cells or infected cells that overexpress the TIGIT ligand, preferably PVR.

[0221] Non-limiting examples of cancer include colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, thyroid cancer, leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloblastic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors (including but not limited to sarcomas and malignant epithelial tumors such as fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor). Leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary epithelial carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. In some embodiments, the infection is a viral, bacterial, fungal, or parasitic infection. In certain specific embodiments, the infection is HIV infection.

[0222] This disclosure also provides cell therapies, and chimeric antigen receptor (CAR) T-cell therapies in some embodiments. Suitable T cells can be used, which are contacted with an anti-TIGIT antibody of this disclosure or a fragment thereof (or optionally engineered to express the anti-TIGIT antibody of this disclosure or a binding fragment thereof). After such contact or engineering, the T cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer disclosed herein. The T cells may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, without limitation. In some embodiments, the T cells are isolated from a cancer patient. In some embodiments, the T cells are provided by a donor or from a cell bank. When the T cells are isolated from a cancer patient, unwanted immune responses can be minimized. When the T cells are provided by a donor other than the patient or from a cell bank, one or more genes encoding the T-cell receptor and HLA genes may be knocked out.

[0223] The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the anti-TIGIT antibody or fragment thereof disclosed herein used, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combinations, and the severity of the specific disease being treated. The judgment of these factors by healthcare professionals is a matter of routine skill in the art. Dosage also depends on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0224] In some embodiments, the antibodies or antigen-binding fragments thereof, bispecific antibodies, peptides, conjugates, compositions, articles, or kits disclosed herein are used in combination with antitumor agents, antiviral agents, antibacterial or antibiotic agents, or antifungal agents. Any of these agents known in the art may be administered in the compositions disclosed herein.

[0225] In another embodiment, the antibodies or antigen-binding fragments thereof, bispecific antibodies, peptides, conjugates, compositions, articles, or kits of this disclosure are administered in combination with chemotherapeutic agents. Chemotherapy agents that can be administered with the compositions of this disclosure include, but are not limited to, antibiotic derivatives (such as doxorubicin, bleomycin, daunorubicin, and actinomycin D); anti-estrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouracil, interferon α-2b, glutamate, pricamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estradiol, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estradiol sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol phosphate). diphosphate, chlorestrin and testosterone); nitrogen mustard derivatives (e.g., melphalan, chlorambucil, dichloromethyldiethylamine (nitrogen mustard) and thiotepa); steroids and combinations thereof (e.g., betamethasone sodium phosphate); and others (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0226] In another embodiment, the antibodies or antigen-binding fragments thereof, bispecific antibodies, peptides, conjugates, compositions, articles, or kits of this disclosure are administered in combination with cytokines, wherein the cytokines include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α. In yet another embodiment, the compositions of this disclosure are administered in combination with other treatment or prophylactic regimens (e.g., radiotherapy).

[0227] The antibodies or antigen-binding fragments thereof disclosed herein, bispecific antibodies, peptides, conjugates, compositions, articles, or kits may be used in some embodiments with immune checkpoint inhibitors. Immune checkpoints are molecules in the immune system that either upregulate (co-stimulatory molecules) or downregulate signaling. Many cancers protect themselves from the immune system by suppressing T cell signaling. Immune checkpoint inhibitors can help block this protective mechanism. Immune checkpoint inhibitors can target any one or more of the following checkpoint molecules: 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; galactoglobulin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; VSIG8.

[0228] Programmed T cell death 1 (PD-1) is a transmembrane protein found on the surface of T cells. When it binds to programmed T cell death ligand 1 (PD-L1) on tumor cells, it leads to the suppression of T cell activity and a reduction in T cell-mediated cytotoxicity. Therefore, PD-1 and PD-L1 are immune downregulation points or immune checkpoint "off switches". Examples of PD-L1 inhibitors include, but are not limited to, nivolumab (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pildizumab, AMP-224, MEDI0680 (AMP-514, PDR001, MPDL3280A, MEDI4736, BMS-936559, and MSB0010718C). Programmed death-ligand 1 (PD-L1), also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. Non-limiting examples of PD-L1 inhibitors include atezolizumab (Tecentriq), durvalumab (MEDI4736), avermab (MSB0010718C), MPDL3280A, and BMS0010718C. 935559 (MDX-105) and AMP-224. CTLA-4 is a protein receptor that downregulates the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675,206). Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint receptor on the cell surface that suppresses immune responses through its action on Tregs and its direct action on CD8+ T cells. LAG-3 inhibitors include, but are not limited to, LAG525 and BMS-986016. CD28 is present in almost all human CD4+ T cells and about half of CD8+ T cells. Constitutive expression on T cells. Promotes T cell proliferation. Non-restrictive examples of CD28 inhibitors include TGN1412. CD122 increases the proliferation of CD8+ effector T cells. Non-restrictive examples include NKTR-214. 4-IBB (also known as CD137) is involved in T cell proliferation. CD137-mediated signaling is known to also protect T cells, especially CD8+ T cells, from activation-induced cell death. PF-05082566, urelumab (BMS-663513), and lipid transporters are examples of CD137 inhibitors.

[0229] For any of the above-mentioned combination therapies, the antibodies or antigen-binding fragments thereof, bispecific antibodies, peptides, conjugates, compositions, products or kits disclosed herein may be administered simultaneously or separately from other anticancer agents.

[0230] In one embodiment, a method for treating or inhibiting an infection in a patient in need is provided, comprising administering to the patient an effective amount of an antibody of the present disclosure or an antigen-binding fragment thereof, a bispecific antibody, a peptide, a conjugate, a composition, an article, or a kit.

[0231] Example

[0232] Example 1. Generation of anti-TIGIT antibody

[0233] BALB / c mice (6 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized twice via intramuscular injection with recombinant human TIGIT / MIgG2aFc protein (self-made, NCBI accession number: NP_776160.2, extracellular domain Met22-Pro141) placed in QuickAntibody-Mouse 5W adjuvant (Beijing Bio-Long Immunotherapy Co., Ltd. #KX0210041). Thirteen days after the second immunization, the mice were given an intraperitoneal booster immunization with TIGIT / MIgG2aFc protein placed in PBS. Three days after booster immunization, spleens were dissected, and spleen cells were fused with P3X63Ag8.653 myeloma cells (cell bank, Chinese Academy of Sciences, #TCM10) using PEG1500 (polyethylene glycol 1500, Roche #783641, 10×4 mL, dissolved in 75 mM Hepes, PEG 50% w / v). Cloning was performed using HAT selection (Sigma #H0262) and HFCS (hybridoma fusion and clonal supplement, 50x, Roche #11-363-735-001). Hybridoma supernatants were screened using ELISA and cell-based assays to produce antibodies that bind to human TIGIT. Selected mouse anti-TIGIT clones were humanized using CDR transplantation and reverse mutagenesis.

[0234] Antibody humanization was achieved through CDR grafting. The acceptor framework was selected. The variable domain sequences of the parent antibody were searched in the human germline database using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast). Five different human acceptors (i.e., human variable regions with high homology to the parent antibody) were selected for each heavy and light chain. The CDRs of the human acceptors were replaced with their corresponding mouse sequences, thus forming the humanized variable domain sequences. The CDR sequences of the heavy and light chains (SEQ ID NO: 1-6) are shown below. Five humanized heavy chains and five humanized light chains were designed, synthesized, and inserted into an expression vector. These humanized antibodies were expressed and then used for affinity ranking tests.

[0235] Example 2: Expression and purification of anti-TIGIT antibody

[0236] DNA sequences encoding the heavy and light chains of humanized IgG were synthesized and inserted into the pTT5 vector (available from Genscript Biotech) to construct a full-length IgG expression plasmid. Expression of the chimeric antibody was performed in Expi 293F cell cultures (available from ThermoFisher Scientific), with the supernatant purified using a protein A affinity column. The purified antibody was exchanged for PBS with buffer using a PD-10 desalting column (available from ThermoFisher Scientific). The concentration and purity of the purified antibody were determined by OD280 and SDS-PAGE, respectively. The humanized antibody was expressed in HEK 293 cell cultures. Cells were centrifuged to precipitate. The supernatant was filtered and analyzed by SDS-PAGE. Figure 1 ).

[0237] Example 3. SPR analysis of the binding affinity between anti-TIGIT antibody and human TIGIT

[0238] To rank antibodies by affinity, antibodies (including those generated in Examples 1 and 2, and chimeric VH+VL (parental mouse VH+VL bound to human Fc)) were immobilized on a sensor chip using an Fc capture method. TIGIT was used as the analyte. The surface was allowed to regenerate before injecting another antibody. This process was repeated until all antibodies were analyzed. The experimental data were locally fitted to a 1:1 interaction model using Biacore 8K evaluation software to obtain the antibody detachment rate. Antibodies were ranked according to their dissociation rate constant (off-rate, kd) (Table 1). Based on the ranking results, the top four clones were selected.

[0239] Table 1. Affinity Measurement Data

[0240]

[0241] The CDR sequences of all antibodies in Table 1 are shown below.

[0242] CDR1H amino acid sequence (SEQ ID NO:1)

[0243] GYTFSRYWIE

[0244] CDR2H amino acid sequence (SEQ ID NO:2)

[0245] EIFPGSGGTNYNEKFKG

[0246] CDR3H amino acid sequence (SEQ ID NO:3)

[0247] HLGALDY

[0248] CDR1L amino acid sequence (SEQ ID NO:4)

[0249] SASSSVSYIH

[0250] CDR2L amino acid sequence (SEQ ID NO:5)

[0251] RTSNLAS

[0252] CDR3L amino acid sequence (SEQ ID NO:6)

[0253] QQYHSNPWT

[0254] Heavy chain variable region (VH2) amino acid sequence (SEQ ID NO:7)

[0255] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGEIFPGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARHLGALDYWGQGTLVTVSS

[0256] Heavy chain variable region (VH3) amino acid sequence (SEQ ID NO:8)

[0257] QVQLVQSGAEVKKPGASVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGEIFPGSGGTNYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHLGALDYWGQGTLVTVSS

[0258] Heavy chain variable region (VH4) amino acid sequence (SEQ ID NO:9)

[0259] EVQLVQSGAEVKKPGESLKISCKGSGYTFSRYWIEWVRQMPGKGLEWMGEIFPGSGGTNYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARHLGALDYWGQGTLVTVSS

[0260] Light chain variable region (VL2) amino acid sequence (SEQ ID NO:10)

[0261] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYIHWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSNPWTFGGGTKLEIK

[0262] Light chain variable region (VL3) amino acid sequence (SEQ ID NO:11)

[0263] EIVLTQSPGTLSLSPGERATLSCSASSSVSYIHWYQQKPGQAPRLLIYRTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHSNPWTFGGGTKLEIK

[0264] Light chain variable region (VL4) amino acid sequence (SEQ ID NO:12)

[0265] DIVMTQSPDSLAVSLGERATINCSASSSVSYIHWYQQKPGQPPKLLIYRTSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYHSNPWTFGGGTKLEIK

[0266] Heavy chain amino acid sequence 1 (HC1) containing VH2 (SEQ ID NO:13, full-length sequence)

[0267] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGEIFPGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARHLGALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain amino acid sequence 2 (HC2) containing VH3 (SEQ ID NO: 14, full-length sequence)

[0268] QVQLVQSGAEVKKPGASVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGEIFPGSGGTNYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHLGALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0269] Heavy chain amino acid sequence 3 (HC3) containing VH4 (SEQ ID NO:15, full-length sequence)

[0270] EVQLVQSGAEVKKPGESLKISCKGSGYTFSRYWIEWVRQMPGKGLEWMGEIFPGSGGTNYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARHLGALDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0271] The light chain amino acid sequence 1 (LC1) containing VL2 (SEQ ID NO:16, full-length sequence)

[0272] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYIHWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSNPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0273] Light chain amino acid sequence 2 (LC2) containing VL3 (SEQ ID NO:17, full-length sequence)

[0274] EIVLTQSPGTLSLSPGERATLSCSASSSVSYIHWYQQKPGQAPRLLIYRTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHSNPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0275] The light chain amino acid sequence 3 (LC3) containing VL4 (SEQ ID NO:18, full-length sequence)

[0276] DIVMTQSPDSLAVSLGERATINCSASSSVSYIHWYQQKPGQPPKLLIYRTSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYHSNPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0277] The heavy chain amino acid sequence containing the parental antibody VH (chimeric VH, SEQ ID NO:19, full-length sequence)

[0278] QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEIFPGSGGTNYNEKFKGKATFTADTSSNTAYMQLTSLTSEDSAVYYCARHLGALDYWGQGTS VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0279] The light chain amino acid sequence containing the parental antibody VL (chimeric VL, SEQ ID NO:20, full-length sequence)

[0280] QIVLTQSPAIMSASPGEKVTISCSASSSVSYIHWYQQKAGSSPKPWIYRTSNLASGVPARLSGSGSGTSYFLTISSMEAEDAATYYCQQYHSNPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0281] For further explanation, the inclusion relationships between the sequences above are shown in Table 2. The sequence on the right is contained within the sequence on the left in the same row.

[0282] Table 2:

[0283]

[0284] Example 4. Combination of ELISA measurement with human TIGIT

[0285] Coat a MaxiSorp 96-well plate (NUNC#449824) with 2 μg / mL human TIGIT / MIgG2aFc protein (prepared, a method well known in the art) in 1×PBS (50 μL / well). Incubate the plate overnight at 4°C. Remove the coating and wash once with 200 μL / well of PBST (1×PBS containing 0.05% Tween-20). Then add 200 μL / well of blocking buffer (1×PBS containing 0.05% Tween-20, 3% BSA) and incubate at room temperature for 1 hour. Remove the blocking buffer and wash three times with 200 μL / well of PBST. Dilute the antibody VH2+VL4 (produced in Example 2) and the human IgG1 isotype control (hIgG1, Sigma#I5154-1MG) with 1×PBS and add to the plate (50 μL / well). Incubate the plate at room temperature for 2 hours. Remove the antibody from the wells and wash the plate three times with 200 μL / well PBST. Dilute goat anti-human IgG (H&L)-HRP secondary antibody (Jackson ImmunoResearch #109-035-088) 1:5000 in 1×PBS and add it to each well (50 μL / well). Incubate the plate at room temperature for 1 hour. Remove the secondary antibody and wash the plate five times with 200 μL / well PBST. Add 50 μL / well TMB (eBioscience #85-00-4201-56) and incubate at room temperature for several minutes. Then add 50 μL / well 2N H2SO4 to stop the reaction. Measure the optical density at 450 nm. The EC50 is 0.47 nM. This result indicates that the anti-TIGIT antibody can bind to human TIGIT with high affinity. Figure 2 ).

[0286] Example 5. Binding with human TIGIT expressed on cells

[0287] DNA encoding full-length human TIGIT (NCBI accession number: NP_776160.2) was cloned into the pcDNA3.4 vector (Invitrogen #A14697) and transfected into Jurkat cells (cell bank, Chinese Academy of Sciences, #TCHU123) by electroporation. Stable cell lines were generated by G418 selection and limiting dilution, and named Jurkat / TIGIT cells.

[0288] Jurkat / TIGIT cells were incubated at 4°C for 30 minutes with different concentrations of anti-TIGIT antibody VH2+VL4 or human IgG1 isotype controls. The cells were then washed once with FACS buffer (PBS with 2% FBS) and incubated at 4°C for 30 minutes with Alexa Fluor 594 AffiniPure goat anti-human IgG secondary antibody (Jackson ImmunoResearch #109-585-088). After washing once with FACS buffer, the cells were resuspended in 200 μL of FACS buffer. The stained cells were analyzed using a BD LSR Fortessa flow cytometer. Figure 3 As shown, the anti-TIGIT antibody VH2+VL4 exhibits a high binding affinity for TIGIT expressed on the cell surface.

[0289] Example 6. Combination with macaque TIGIT

[0290] DNA encoding full-length rhesus monkey TIGIT (NCBI accession number: XP_015300911.1) was cloned into the pcDNA3.4 vector (Invitrogen #A14697) and transfected into 293T cells (cell bank, Chinese Academy of Sciences, #SCSP-502) using polyethyleneimine Max reagent (Polysciences #24765-2). Forty-eight hours post-transfection, the 293T cells expressing rhesus monkey TIGIT were incubated with different concentrations of biotin-labeled (Thermo #21338) anti-TIGIT antibody VH2+VL4 at 4°C for 20 minutes. The cells were then washed once with FACS buffer (PBS with 2% FBS) and incubated with Brilliant Violet 421 streptavidin (Biolegend #405225) at 4°C for 30 minutes. After washing once with FACS buffer, the cells were resuspended in 200 μL of FACS buffer. The stained cells were analyzed using a BD FACS Celesta flow cytometer. Figure 4 As shown, the anti-TIGIT antibody VH2+VL4 exhibits a high binding affinity for macaque TIGIT.

[0291] Example 7. Functional determination of anti-TIGIT antibody in primary T cells

[0292] 96-well flat-bottom plates (NUNC#167008) were coated with anti-human CD3 antibody (0.1 μg / mL, BD Pharmingen#555329) and human CD155 protein (0.5 μg / mL, Sino Biological#10109-H02H) and incubated overnight at 4°C. The next day, PBMCs were labeled with CFSE (Sigma#21888-25MG) and seeded into the pre-coated wells (2 × 10⁻⁶). 5 Cells per well (each well containing different concentrations of anti-TIGIT antibody VH2+VL4, tiragolumab monoclonal antibody (produced in Example 7), or hIgG1 isotype control). The plates were then incubated in a CO2 incubator for 72 hours. After 72 hours, the cells were transferred to a 96-well U-shaped plate (NEST#701101) for staining. Cells were then stained with Fixable ViabilityDye eFluor diluted in PBS. TM Cells were incubated with 660 (Invitrogen #65-0864-14) at 4°C for 15 minutes. Fluorescently labeled antibody mixtures were prepared in FACS buffer as described below: Alexa Fluor 700 mouse anti-human CD3 (BD ​​Pharmingen #557943), PE-CF594 mouse anti-human CD4 (BD Pharmingen #562402), and BV421 mouse anti-human CD8 (BDPharmingen #562428). Cells were then incubated with the antibody mixture at 4°C for 30 minutes. After one wash, cells were analyzed using a BDFACS Celesta flow cytometer. Cell proliferation was measured using a dye diluent from CFSE.

[0293] like Figure 5A and 5B As shown, CD155 inhibits the proliferation of CD4+ and CD8+ T cells. The anti-TIGIT antibody VH2+VL4 can reverse the CD155-induced inhibition in a dose-dependent manner by blocking TIGIT expression on T cells. Compared to tiragolumab, the anti-TIGIT antibody VH2+VL4 exhibits stronger competitive inhibition of CD155.

[0294] Example 8. In vivo animal studies of antitumor activity

[0295] Antibody expression and purification for animal studies

[0296] DNA sequences encoding VH2 (SEQ ID NO:13) and VL4 (SEQ ID NO:18) were subcloned into the pcDNA3.4 vector (Invitrogen#A14697) to construct two plasmids, pcDNA3.4-VH2 and pcDNA3.4-VL4. pcDNA3.4-VH2 and pcDNA3.4-VL4 were prepared using the endotoxin-free plasmid DNA Maxiprep kit (TIANGEN#DP117). Antibody expression was performed in 293-F cells (Invitrogen#R79007). The antibody in the culture supernatant was purified using a protein A affinity column (Yeasen#36410ES08). The purified antibody was dialyzed and buffer-exchanged to histidine buffer (20 mM histidine, 5% sucrose, 0.02% Tween 80, pH 5.5). The concentration and purity of the purified antibody were determined by OD280 and SDS-PAGE, respectively. The positive control antibody, tiragolumab (CAS#1918185-84-8), was expressed and purified using the same method.

[0297] Animal research

[0298] In this study, the antitumor activity of antibody VH2+VL4 was investigated using a human TIGIT knock-in mouse tumor model carrying CT26.

[0299] Mouse colon cancer cells CT26 (cell bank, Chinese Academy of Sciences, #TCM37) were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin. 5×10⁻⁶ cells were placed in 100 μL of PBS. 5 One CT26 cell was injected subcutaneously into the right dorsal side of each human TIGIT gene knock-in mouse (BALB / c, female, 6-8 weeks old, GemPharmatech). When the average tumor volume reached approximately 63 mm... 3 Mice were randomly divided into groups of eight and administered antibodies. On days 8, 11, 14, and 17, mice were intraperitoneally injected with anti-TIGIT antibody VH2+VL4 and the positive control antibody tiragolumab at a dose of 10 mg / kg. Control mice were injected with histidine buffer (solvent). Tumors were measured every two days using calipers. Tumor volume was calculated using the following formula: width... 2 × Length / 2 (mm) 3 When the average tumor volume of any group reaches 2000 mm... 3 At that time, the mice were euthanized.

[0300] like Figure 6As shown, the anti-TIGIT antibody VH2+VL4 exhibited strong inhibitory effects on tumor growth in vivo, comparable to tiragolumab monoclonal antibody. No significant weight changes were associated with antibody administration. sequence list <110> Suzhou Xinkanghe Biomedical Technology Co., Ltd. Beijing Xinkanghe Biomedical Technology Co., Ltd. <120> Anti-TIGIT antibodies and their uses <130> PF02115 <150> PCT / CN2021 / 094434 <151> 2021-05-18 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1H <400> 1 Gly Tyr Thr Phe Ser Arg Tyr Trp Ile Glu 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2H <400> 2 Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 3 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR3H <400> 3 His Leu Gly Ala Leu Asp Tyr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1L <400> 4 Ser Ala Ser Ser Ser Val Ser Tyr Ile His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR2L <400> 5 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR3L <400> 6 Gln Gln Tyr His Ser Asn Pro Trp Thr 1 5 <210> 7 <211> 116 <212> PRT <213> Artificial sequence <220> <223> VH2 <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH3 <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 9 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH4 <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 106 <212> PRT <213> artificial sequence <220> <223> VL2 <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> VL3 <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 106 <212> PRT <213> artificial sequence <220> <223> VL4 <400> 12 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu 65 70 75 80 Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 446 <212> PRT <213> artificial sequence <220> <223> HC1 <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 14 <211> 446 <212> PRT <213> Artificial sequence <220> <223> HC2 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 15 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> HC3 <400> 15[[ID=A]] Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 16 <211> 213 <212> PRT <213> Artificial sequence <220> <223> LC1 <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 17 <211> 213 <212> PRT <213> Synthetic Sequence <220> <223> LC2 <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys [[ID=~10]]130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 18 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> LC3 <400> 18 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu 65 70 75 80 Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 19 <211> 446 <212> PRT <213> Synthetic sequence <220> <223> HC containing the parental antibody VH <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Phe Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Leu Gly Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 20 <211> 213 <212> PRT <213> artificial sequence <220> <223> Contains the LC of VL <400> 20 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Tyr Gln Gln Lys Ala Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Leu Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Phe Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Asn Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210

Claims

1. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to the extracellular domain of TIGIT, and its binding affinity is determined to be better than 10 nM by SPR analysis, wherein... (a) The HC contains CDR1H, wherein the amino acid sequence of CDR1H is GYTFSRYWIE (SEQ ID NO:1), CDR2H, wherein the amino acid sequence of CDR2H is EIFPGSGGTNYNEKFKG (SEQ ID NO:2), and CDR3H, wherein the amino acid sequence of CDR3H is HLGALDY (SEQ ID NO:3); (b) The LC includes CDR1L, wherein the amino acid sequence of CDR1L is SASSVSYIH (SEQ ID NO:4), CDR2L, wherein the amino acid sequence of CDR2L is RTSNLAS (SEQ ID NO:5), and CDR3L, the amino acid sequence of which is QQYHSNPWT (SEQ ID NO:6).

2. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a human receptor framework.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the HC variable region sequence comprises an amino acid sequence selected from: SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from: SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the LC variable region sequence comprises an amino acid sequence selected from SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:

12.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein... 1) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:11; 2) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:8; and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:12; 3) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:7; and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:12; or 4) The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:9; and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:

10.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:

12.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is an IgG isotype.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment comprises any one of the following: Fab, F(ab')2, Fab', scFv, Fv, Fd, dAb and bisomatic antibodies.

10. A bispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 and a second antibody or antigen-binding fragment thereof.

11. The bispecific antibody of claim 10, wherein the second antibody or its antigen-binding fragment specifically binds to a tumor antigen expressed on the surface of tumor cells or an immune checkpoint protein expressed on the surface of immune cells or tumor cells, wherein the tumor antigen or the immune checkpoint protein comprises any one of the following: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD2 7; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; Human Papillomavirus-E6; Human Papillomavirus-E7; JAM-3; KID3; KID31; KSA; LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosyltransferase; tumor suppressor M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; VEGFR; 2B4; 4-1BB; 4-1BB ligand; B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; C D19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; galactolectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; and VSIG8.

12. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9, wherein a therapeutic agent is attached.

13. The conjugate according to claim 12, wherein the therapeutic agent is a cytotoxin or a radioisotope.

14. A composition comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-9, a bispecific antibody as claimed in claim 10 or 11, or a conjugate as claimed in claim 12 or 13, and a pharmaceutically acceptable excipient.

15. Lymphocytes derived from a subject and subjected to in vitro treatment with an antibody or antigen-binding fragment thereof as described in any one of claims 1-9.

16. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

17. An expression vector comprising the nucleic acid as described in claim 16.

18. A polypeptide comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-9.

19. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9, the bispecific antibody as described in claim 10 or 11, the conjugate as described in claim 12 or 13, the composition as described in claim 14, the lymphocyte as described in claim 15, or the polypeptide as described in claim 18 in the preparation of a medicament for treating cancer in a subject; in, The HC variable region sequence comprises the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having an identity greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of SEQ ID NO:12; The cancers mentioned therein are selected from one or more of the following: lymphoma, melanoma, colorectal adenocarcinoma, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, stomach cancer, and clear cell renal cell carcinoma.

20. The use according to claim 19, wherein the antibody or its antigen-binding fragment, bispecific antibody, polypeptide, conjugate, composition, or lymphocyte is administered in combination with one or more antibodies or antibody fragments or anticancer agents, wherein the antibody or antibody fragment or anticancer agent is selected from any one or more of the following: antibodies against checkpoint molecules or their receptors, anti-epidermal growth factor receptor agents, EGFR tyrosine kinase inhibitors, alkylating agents, and topoisomerase inhibitors.

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