Anti-TIGIT antibodies and their applications

Abstract

The present application provides anti-TIGIT (VSIG9, VSTM3, WUCAM) antibodies and their uses. The present application also provides chimeric antibodies and humanized antibody forms of anti-TIGIT antibodies. The anti-TIGIT antibodies of the present application can specifically bind to mammalian PVR, preferably have a high affinity for human and / or primate TIGIT proteins, can block the binding of TIGIT to its ligand at a certain concentration, regulate receptor activity, stimulate T cell activation, inhibit Treg function, thereby better exerting the effects of effector T cells, etc. The anti-TIGIT antibodies of the present application can be used to treat TIGIT-related diseases.

Description

Technical Field

[0001] The present application relates to the field of immunology, and more specifically, to anti-TIGIT antibodies and applications thereof. Background Art

[0002] The concept of the immune system identifying and eliminating cancer cells was first proposed more than 100 years ago. T cell reactivity to cancer-related antigens can be detected in the blood of patients with different types of cancer. In the effective anti-tumor immune process, T cells, as the core executors, are first activated by antigen recognition signals mediated by T cell receptors (TCRs). At the same time, numerous co-stimulatory and co-inhibitory signals finely regulate the intensity and quality of T cell responses. These inhibitory signals are immune checkpoints.

[0003] The immune system has evolved multiple mechanisms to prevent harmful activation of immune cells. One of these mechanisms is the balance between T cell co-stimulatory and co-inhibitory signals. The B7-1 (CD80) / B7-2 (CD86)-CTLA-4 pathway is the most characterized inhibitory pathway for T cell activation. Another inhibitory pathway involves programmed death-1 (PD-1), which negatively regulates T cell activation by interacting with PD-L1 (B7-H1) and PD-L2 (B7-DC). The third co-inhibitory molecule of T cell activation, B cell and T cell attenuator (BTLA), is a cell surface molecule similar to CTLA-4 and PD-1. Because these inhibitory co-receptors inhibit T cell proliferation and cytokine production, they are believed to play an important role in maintaining immune homeostasis and tolerance. A large amount of evidence indicates that co-stimulatory molecules play a key role in preventing autoimmune diseases, because the loss of these molecules or functional mutations accelerate genetic autoimmunity and polymorphisms are associated with human genetic susceptibility to autoimmune diseases.

[0004] Under physiological conditions, co-stimulatory molecules maintain a balance with immune checkpoint molecules, thereby minimizing damage to surrounding normal tissues, maintaining tolerance to one's own tissues, and avoiding autoimmune reactions. Tumor cells can use this mechanism to abnormally upregulate co-inhibitory molecules and their related ligands, inhibit T cell activation, and thus escape immune killing. Blocking immune checkpoints is one of the effective strategies to enhance T cell activation and is also a hot target for the development of anti-tumor drugs in recent years. So far, three immune checkpoint inhibitors have been approved in the United States for the treatment of various types of cancer, targeting CTLA-4 and PD-1 / PD-L1.

[0005] In 2008, Xin Yu et al. (2009) used a gene chip scanning retrieval strategy to sequence activated human T cells and further studied some protein molecules with immunoregulatory-like domains. They discovered a new molecule expressed on T cells and NK cells. The molecule has an immunoglobulin-like domain, a transmembrane region and an immunoreceptor protein tyrosine inhibitory motif (ITIM), so it was named TIGIT (T cell immunoglobulin and ITIM domain).

[0006] Blocking immune checkpoint molecules mainly refer to the co-inhibitory signal molecules of T cell activation - T cell co-inhibitory receptors. Studies have shown that TIGIT, CTLA-4 and PD-1 play an immunosuppressive role in the process of T cell activation, thereby inhibiting the immune killing function of T cells against tumor cells; and blocking monoclonal antibodies targeting these three targets can relieve this immunosuppression and restore the anti-tumor immune function of T cells.

[0007] The TIGIT gene is located on human chromosome 16 and encodes a type I transmembrane protein composed of 244 amino acids. The human TIGIT molecule has an extracellular region of 141 amino acids, which has an immunoglobulin V-like domain; a transmembrane region of 23 amino acids; and a shorter cytoplasmic region of 80 amino acids, which has a PDZ binding domain and an ITIM motif. The structure of the human TIGIT molecule is as follows Fig.13 As shown. TIGIT (also known as VSIG9, VSTM3, WUCAM) is a member of the immunoglobulin poliovirus receptor family CD28 family-like receptors. It is expressed in T cells and natural killer (NK) cell subsets, but generally, its expression level is very low. When these cells are activated, its protein level will be upregulated. For example, in the tumor microenvironment, TIGIT in tumor-infiltrating lymphocytes is often at a high expression level. TIGIT can bind to receptors CD155 (poliovirus receptor-PVR), CD112 (PVRL2, nectin-2), and CD113 (PVRL3, Nectin-3) in immune cells, non-immune cells, and tumor cells. The binding leads to T cell activation and the cytotoxicity of T cells and natural killer cells is inhibited.

[0008] Specifically, TIGIT can inhibit lymphocytes through three different mechanisms of action: 1) After binding to PVR, TIGIT can send signals through the ITIM and / or ITT motifs in its intracellular tail, thereby inhibiting the killing of tumor cells by NK, CD8+T and other cells; 2) TIGIT can induce PVR signaling in adjacent dendritic cells or tumor cells by binding to PVR, thereby releasing cytokines such as IL-10 and TGF-β, promoting Treg cell activation and thus inhibiting T cell activity; 3) TIGIT can inhibit CD226 signaling by binding to PVR with higher affinity or disrupting CD226 homodimerization.

[0009] Therefore, there is still a need in the art to develop TIGIT-targeting antibodies. Summary of the invention

[0010] To solve the above technical problems, the present application provides an anti-TIGIT antibody and its application. Specifically, the present application provides the following technical solutions.

[0011] In the first aspect, the present application provides an antibody or an antigen-binding portion thereof that specifically binds to TIGIT, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises any one or more of HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GFTFSNYW (SEQ ID NO: 1), the HCDR2 sequence is IRLKSNNYAT (SEQ ID NO: 2), and the HCDR3 sequence is ARLYYGNYFDY (SEQ ID NO: 3) or TRLYYGNYFDY (SEQ ID NO: 4).

[0012] In some embodiments, the antibody or antigen-binding portion thereof further comprises a light chain variable region, which comprises any one or more of LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1 sequence is ENTYSY (SEQ ID NO: 5), the LCDR2 sequence is NAK (SEQ ID NO: 6), and the LCDR3 sequence is QHHYAFSYT (SEQ ID NO: 7).

[0013] In some embodiments, the antibody is a murine antibody. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9.

[0014] In some embodiments, the antibody is a chimeric antibody. Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:10, and / or the amino acid sequence of the light chain is as shown in SEQ ID NO:11.

[0015] In some embodiments, the antibody is a humanized antibody. Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 12 or SEQ ID NO: 13, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0016] In some embodiments, the antibody or antigen binding portion thereof that specifically binds to TIGIT can specifically bind to primate TIGIT.

[0017] In some embodiments, the antibody that specifically binds TIGIT is a monoclonal antibody.

[0018] In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to TIGIT is expressed in 1×10 -9 Up to 1×10 -10 The KD of M specifically binds to the TIGIT molecule.

[0019] In some embodiments, the antibody or antigen-binding portion thereof that specifically binds to TIGIT has TIGIT inhibitor function.

[0020] In some embodiments, the antibody or antigen binding portion thereof that specifically binds to TIGIT is capable of stimulating the activation and proliferation of T cells.

[0021] In a second aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect and a pharmaceutically acceptable carrier.

[0022] In some embodiments, the pharmaceutical composition further comprises one or more other active ingredients. In some embodiments, the active ingredient is a chemotherapeutic agent, a PD-1 binding antagonist, etc.

[0023] In a third aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect.

[0024] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0025] In the fifth aspect, the present application provides a host cell, which comprises the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect.

[0026] In a sixth aspect, the present application provides a vaccine comprising the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect, and an optional immune adjuvant.

[0027] In the seventh aspect, the present application provides the use of the antibody or its antigen-binding portion that specifically binds to TIGIT as described in the first aspect, the pharmaceutical composition as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the vaccine as described in the sixth aspect in the preparation of a drug for inhibiting Treg function, killing cells expressing TIGIT, inducing T cell-mediated responses, improving the function of effector T cells, improving the function of memory T cells, and / or effectively inhibiting tumor growth.

[0028] In the eighth aspect, the present application provides the use of the antibody or its antigen-binding portion that specifically binds to TIGIT as described in the first aspect, the pharmaceutical composition as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the vaccine as described in the sixth aspect in the preparation of a medicament for preventing and / or treating TIGIT-related diseases, such as tumors.

[0029] In some embodiments, the tumor is selected from colon cancer, melanoma, mesocortical tumor, renal cell carcinoma, lymphoma, advanced solid tumors, and metastases thereof.

[0030] In a ninth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect.

[0031] In the tenth aspect, the present application provides a method for preventing and / or treating TIGIT-related diseases, such as tumors, comprising administering to an individual in need the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect, the pharmaceutical composition as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the vaccine as described in the sixth aspect.

[0032] In some embodiments, the method further comprises co-administering other therapeutic agents, such as chemotherapeutic agents, PD-1 binding antagonists, etc.

[0033] The antibody or antigen-binding portion thereof that specifically binds to TIGIT of the present application can specifically bind to TIGIT and has one or more of the following effects: having TIGIT inhibitor function, stimulating T cell proliferation and activation, inducing TIGIT-mediated anti-tumor immune response, and / or inhibiting tumor growth, etc.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The ability of the murine anti-TIGIT antibody of the present application (antibody secreted by hybridoma 18A12) to block the binding of PVR to human TIGIT was demonstrated.

[0036] Figure 2 The binding affinity of the murine anti-TIGIT antibody of the present application (antibody secreted by hybridoma 18A12) to cynomolgus monkey TIGIT is shown.

[0037] Figure 3 The chimeric anti-TIGIT antibody (IgG1 subtype) of the present application has a binding affinity to human TIGIT.

[0038] Figure 4 The efficacy experiment of the chimeric anti-TIGIT antibody (IgG1 subtype) and anti-PD-1 antibody of the present application in inhibiting the MC38 colon cancer model in B-hTIGIT mice is shown. A: the change of tumor volume of each group of mice; B: the change of mouse body weight with the administration time; C, D and E: the specific tumor inhibition data of the three groups; F: the tumor photographed after the mice were killed; G: the calculated tumor weight / body weight data.

[0039] Figure 5 The binding affinities of the six anti-TIGIT humanized antibodies (IgG1 subtype) of the present application to human TIGIT are shown, which were detected by flow cytometry.

[0040] Figure 6 The binding affinities of the six anti-TIGIT humanized antibodies (IgG1 subtype) of the present application to cynomolgus monkey TIGIT are shown, which were detected by flow cytometry.

[0041] Figure 7 The ability of the six anti-TIGIT humanized antibodies (IgG1 subtype) of the present application to block the binding of PVR to human TIGIT was demonstrated, which was detected by flow cytometry.

[0042] Figure 8 The pharmacodynamics experimental results of the chimeric anti-TIGIT antibody (IgG1 subtype) of the present application in the MC38 model are shown. A: mouse tumor volume change diagram; B: mouse body weight change diagram; C and D: tumor volume change diagrams of two groups of mice; E and F: tumor spleen display diagrams after mouse dissection; G and H: spleen weight ratio to tumor weight ratio analysis diagram (NS represents the saline solvent control group).

[0043] Fig. 9 The pharmacodynamic experimental results of the chimeric anti-TIGIT antibody (IgG1 subtype) of the present application in the CT26 model are shown: a trend chart of the changes in tumor volume and body weight of mice after administration (NS represents the saline solvent control group).

[0044] Fig.10The pharmacodynamic experimental results of the chimeric anti-TIGIT antibody (IgG1 subtype) of the present application in the CT26 model are shown: individual tumor growth trend graph of each group of mice after administration.

[0045] Fig.11 The pharmacodynamic experimental results of the chimeric anti-TIGIT antibody (IgG1 subtype) of the present application in the CT26 model are shown: the anatomical diagrams of the tumors and spleens of mice in each group after the mice were killed when the tumor volume reached the humane endpoint.

[0046] Fig.12 The pharmacodynamic experimental results of the chimeric anti-TIGIT antibody (IgG1 subtype) of the present application in the CT26 model are shown: after the tumor volume reached the humane endpoint and the mice were killed, the tumors and spleens of each group of mice were weighed and then calculated and analyzed to obtain the tumor-to-weight ratio and spleen weight ratio analysis graphs.

[0047] Fig.13 A schematic diagram of the structure of the human TIGIT molecule is shown, in which the signal peptide is amino acids 1-21; the extracellular segment is amino acids 22-141 (including the IgV region: amino acids 22-124); the transmembrane region is amino acids 142-162; the intracellular segment is amino acids 163-244 (including the ITIM motif: amino acids 229-234, as marked by the black box in the figure).

[0048] Detailed Description of the Invention

[0049] The following definitions and methods are provided to better define the present application and guide those of ordinary skill in the art in the practice of the present application. Unless otherwise specified, the terms of the present application are understood according to conventional usage by those of ordinary skill in the relevant art.

[0050] definition

[0051] To facilitate understanding of this application, certain terms used herein are first defined.

[0052] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, i.e., two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). The heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region comprises three domains, i.e., CH1, CH2, and CH3. The light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). In some embodiments, from N-terminus to C-terminus, both the light chain and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0053] As used herein, the term "antigen binding portion" of an antibody refers to a portion or segment of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding portion may include a heavy chain variable region (VH), a light chain variable region (VL), or both. The antigen binding portion of an antibody can be prepared from a complete antibody molecule using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, etc. Non-limiting examples of antigen binding portions include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and the minimum recognition unit (e.g., isolated CDR) composed of amino acid residues that mimic the hypervariable region of an antibody. The term "antigen binding portion" also includes other engineered molecules, such as double antibodies, three antibodies, four antibodies, and mini antibodies, etc. For example, the Fd fragment described herein refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of VL and VH domains in a single arm of an antibody; and the dAb fragment (Ward et al., Nature 1989; 341: 544-546) consists of a VH domain.

[0054] It is well known to those skilled in the art that the complementarity determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR sequence of VH or VL, namely the Kabat definition and the Chothia definition, for example, see Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR region sequence in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiments of the present application, the Kabat definition of CDR sequences is used. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively.

[0055] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0056] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, such as the ability of an antibody to bind to a specific antigen with an affinity at least twice greater than its affinity for a non-specific antigen. However, it should be understood that an antibody is capable of specifically binding to two or more antigens related to its sequence. For example, the antibodies of the present invention can specifically bind to TIGIT in humans and non-humans (e.g., mice or non-human primates).

[0057] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for naturally occurring mutations that may exist in a small number of individuals. The monoclonal antibodies described herein particularly include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and the remainder of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, as long as they can exhibit the desired biological activity (see, U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

[0058] As used herein, the term "murinized antibody" refers to any antibody in which all constant domain sequences are mouse sequences. Such antibodies can be produced by hybridomas.

[0059] As used herein, the term "chimeric antibody" refers to an antibody comprising segments from two or more different antibodies. In some embodiments, one or more CDRs are derived from mouse anti-TIGIT antibodies. In other embodiments, all CDRs are derived from mouse anti-TIGIT antibodies. In some embodiments, CDRs from more than one mouse anti-TIGIT antibody are combined in a chimeric antibody. For example, a chimeric antibody may include CDR1 from a light chain in a first mouse anti-TIGIT antibody, CDR2 from a light chain in a second mouse anti-TIGIT antibody, and CDR3 from a light chain in a third mouse anti-TIGIT antibody, and CDRs from a heavy chain may be derived from one or more other anti-TIGIT antibodies. In addition, the framework region may be from the same anti-TIGIT antibody or from one or more different individuals. In some embodiments, the chimeric antibodies described herein include variable regions of murine antibodies (including heavy chain variable regions VH and / or light chain variable regions VL) and constant regions of human antibodies.

[0060] As used herein, the term "humanized antibody" refers to a CDR-transplanted antibody, specifically an antibody produced by transplanting the CDR region sequence of other species, such as mice, into the antibody variable region framework of a human. The purpose is to overcome the strong immune side effects induced in the human body by chimeric antibodies due to carrying a large number of protein components of other species, such as mice.

[0061] As used herein, the term "nucleic acid molecule" may refer to a DNA molecule and an RNA molecule, which may be single-stranded or double-stranded. The nucleic acid molecule may also be a cDNA.

[0062] As used herein, the term "TIGIT-related disease" includes diseases and / or symptoms associated with the TIGIT signaling pathway. Exemplary TIGIT-related diseases or conditions include tumors, such as colon cancer, melanoma, mesocortical tumors, renal cell carcinoma, lymphoma, advanced solid tumors, and metastatic tumors of the above.

[0063] As used herein, the term "EC50" refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that can cause 50% of the maximal effect.

[0064] As used herein, the term "immune response" refers to a biological response in vertebrates to foreign agents that protects the organism against such agents and diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by any of these cells or by the liver (including antibodies, cytokines, and complement), which results in selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancerous or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation from the vertebrate body. The immune response includes T cells (e.g., effector T cells) or Th cells (e.g., CD4 + or CD8 + T cells) or the inhibition of Treg cells.

[0065] As used herein, the term "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0066] As used herein, the term "treatment" refers to any type of intervention or method performed on a subject or the administration of an active agent thereto, wherein the object is to reverse, alleviate, ameliorate, inhibit or relieve or prevent the progression, development, severity or recurrence of symptoms, complications, conditions or disease-related conditions.

[0067] As used herein, the term "prevention" refers to administration to a subject not suffering from a disease to prevent the occurrence of the disease or to minimize its effects, if any. DETAILED DESCRIPTION

[0068] The inventors screened and obtained a hybridoma cell line, the antibody in the supernatant of which can bind to human or monkey TIGIT and can block the binding of human PVR to TIGIT.

[0069] The inventors also prepared chimeric antibodies and humanized antibody forms from mouse anti-TIGIT antibodies through genetic engineering. These antibody forms can also bind to human or monkey TIGIT and block the binding of human PVR to TIGIT, thereby effectively inducing TIGIT-mediated immune responses and playing a role in preventing or treating TIGIT-related diseases.

[0070] The present application also provides nucleic acid molecules encoding the antibody or its antigen-binding fragment, expression vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or expression vectors, methods for preparing and purifying the antibody, and medical and biological applications of the antibody or its antigen-binding fragment, such as preventing or treating TIGIT-related diseases or conditions. The present application also covers methods for detecting TIGIT and regulating TIGIT activity using the antibody or its antigen-binding fragment, as well as related detection reagents or kits.

[0071] Suitable techniques that can be used in the antibody method include TIGIT-based affinity purification, native gel purification, HPLC or RP-HPLC, purification on a protein A column, or any combination of these techniques. TIGIT antibody isotype can be determined using an ELISA assay, for example, human Ig can be identified using anti-human Ig adsorbed with mouse Ig.

[0072] TIGIT suitable for producing antibodies can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Forms of TIGIT suitable for producing an immune response include TIGIT subsequences (e.g., immunogenic fragments). Additional forms of TIGIT include TIGIT-expressing cells, TIGIT-containing preparations or cell extracts or fractions, and partially purified TIGIT.

[0073] In the first aspect, the present application provides an antibody or an antigen-binding portion thereof that specifically binds to TIGIT, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises any one or more of HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GFTFSNYW (SEQ ID NO: 1), the HCDR2 sequence is IRLKSNNYAT (SEQ ID NO: 2), and the HCDR3 sequence is ARLYYGNYFDY (SEQ ID NO: 3) or TRLYYGNYFDY (SEQ ID NO: 4).

[0074] In a preferred embodiment, the heavy chain variable region of the antibody or antigen binding portion thereof described herein comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0075] In some embodiments, the antibodies or antigen-binding portions thereof described herein further comprise a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is ENTYSY (SEQ ID NO: 5), the LCDR2 sequence is NAK (SEQ ID NO: 6), and the LCDR3 sequence is QHHYAFSYT (SEQ ID NO: 7).

[0076] In a preferred embodiment, the light chain variable region of the antibody or antigen binding portion thereof described herein comprises LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:7.

[0077] In a preferred embodiment, the antibody or antigen-binding portion thereof described herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 sequence shown in SEQ ID NO:3 or SEQ ID NO:4, and the light chain variable region comprises LCDR1 shown in SEQ ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 sequence shown in SEQ ID NO:7.

[0078] In some more specific embodiments, the antibodies disclosed herein can be anti-TIGIT monoclonal antibodies. Anti-TIGIT antibody types and subtypes can be determined by any method known in the art. Generally, antibody types and subtypes can be determined using antibodies specific for specific antibody types and subtypes. Anti-TIGIT antibody isotypes can be determined using ELISA assays, for example, anti-human Ig adsorbed with mouse Ig can be used to identify human Ig.

[0079] In some embodiments, the antibody described herein is a murine antibody, preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9.

[0080] In a preferred embodiment, the murine antibody described herein comprises a heavy chain variable region as shown in SEQ ID NO:8 and a light chain variable region as shown in SEQ ID NO:9.

[0081] In some embodiments, the antibodies described herein are chimeric antibodies. The chimeric antibodies described herein comprise the variable region of a murine antibody (including the heavy chain variable region VH and / or the light chain variable region VL) and the constant region of a human antibody.

[0082] In a preferred embodiment, the chimeric antibodies described herein comprise the variable regions of a murine antibody (including a heavy chain variable region and a light chain variable region) and the constant regions of a human antibody.

[0083] Preferably, the chimeric antibody comprises the heavy chain shown in SEQ ID NO:10, and / or the light chain shown in SEQ ID NO:11.

[0084] In a preferred embodiment, the chimeric antibody described herein comprises the heavy chain shown in SEQ ID NO:10 and the light chain shown in SEQ ID NO:11.

[0085] In some embodiments, the antibodies described herein are humanized antibodies. The humanized antibodies described herein comprise the CDR regions of murine antibodies (including any one or more of HCDR1, HCDR2, and HCDR3 and / or any one or more of LCDR1, LCDR2, and LCDR3), the framework regions of human antibody variable regions (including any one or more of FR1, FR2, FR3, and FR4), and optionally the constant regions of human antibodies.

[0086] In a preferred embodiment, the humanized antibodies described herein comprise the CDR regions (including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of a murine antibody, the framework regions (including FR1, FR2, FR3 and FR4) of a human antibody variable region, and optionally the constant region of a human antibody.

[0087] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:12 or SEQ ID NO:13, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0088] In a preferred embodiment, the humanized antibody described herein comprises a heavy chain variable region shown in SEQ ID NO:12 or SEQ ID NO:13, and a light chain variable region shown in SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0089] The antibodies described herein may also include mouse or human antibody constant regions. Mouse antibody constant regions include the heavy chain constant regions of mouse IgG1, IgG2a, IgG2b or IgG3 and the κ or λ type light chain constant regions, etc. Human antibody constant regions include the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 and the κ or λ type light chain constant regions, etc.

[0090] In some embodiments, the TIGIT described herein is a primate TIGIT. Preferably, the primate TIGIT described herein is selected from human TIGIT or monkey TIGIT.

[0091] In some embodiments, the antigen binding portion described herein is selected from: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, or a single domain antibody.

[0092] The term "Fab fragment" as used herein comprises a light chain as well as the CH1 and variable regions of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0093] The term "Fab' fragment" as used herein contains a light chain and a portion or fragment of a heavy chain, said portion or fragment containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0094] The term "F(ab')2 fragment" as used herein contains two light chains and two heavy chains, wherein the heavy chain contains a portion of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments, and the two Fab' fragments are linked together by a disulfide bond between the two heavy chains.

[0095] As used herein, the term "Fv fragment" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0096] The term "single-chain Fv" or "scFv" as used herein refers to an antibody fragment comprising the VH domain and the VL domain of an antibody, wherein these domains are present in the form of a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form a desired structure for antigen binding.

[0097] The term "single domain antibody" as used herein refers to an antigen-binding portion comprising a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. It was originally an antibody that was naturally lacking light chains and was discovered in the peripheral blood of alpacas. Although this antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, it is not as easy to stick to each other or even aggregate into clumps as artificially modified single-chain antibody fragments (scFv). More importantly, the VHH structure that is cloned and expressed separately has a structural stability and antigen-binding activity comparable to that of the original heavy chain antibody, and is the smallest unit known to bind to the target antigen. The molecular weight of the VHH crystal is only 15KDa, so it is also called a nanobody (Nanobody, Nb)

[0098] In a second aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to the first aspect and a pharmaceutically acceptable carrier.

[0099] Pharmaceutically acceptable carriers or diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10 ) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C 12 Alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical preparations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the pharmaceutical preparations.

[0100] In some embodiments, the pharmaceutical compositions described herein further comprise one or more other active ingredients, such as agents for treating TIGIT-related diseases such as tumors, such as chemotherapeutic agents and / or PD-1 binding antagonists.

[0101] In a third aspect, the present application provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof described in the first aspect.

[0102] In preferred embodiments, nucleic acid as described herein can be a codon-optimized nucleic acid suitable for expression in a host cell. For example, according to the degeneracy of codons, it still encodes the same protein. The method for codon optimization according to the host cell used is well known to those skilled in the art.

[0103] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0104] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from Escherichia coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages. The example of a vector that can be used for yeast is the 2μ plasmid. Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from functional mammalian vectors (such as those described above) and combinations of functional plasmids and phage DNA.

[0105] Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern & P. ​​Berg, J. Mol. Appl. Genet, 1:327-341 (1982); Subramani et al., Mol. Cell. Biol, 1:854-864 (1981); Kaufinann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol, 159:601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol, 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci. USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), which are incorporated herein by reference in their entireties).

[0106] The expression vector that can be used for the present application contains at least one expression control sequence, which is operably connected to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter region of bacteriophage λ, the control region of the fd coat protein, the glycolytic promoter of yeast, such as the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, such as Pho5, the promoter of yeast α-mating factor, and promoters derived from polyoma viruses, adenoviruses, retroviruses and simian viruses, such as the early and late promoters of SV40 and other sequences known to control the gene expression of prokaryotic or eukaryotic cells and their viruses or their combinations.

[0107] In the fifth aspect, the present application provides a host cell, which comprises the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect.

[0108] In some embodiments, the host cells described herein are mammalian cells. Mammalian cells may include, but are not limited to, CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select suitable host cells as needed.

[0109] The preparation method of the anti-TIGIT monoclonal antibody disclosed herein may include: culturing host cells under expression conditions to express the anti-TIGIT monoclonal antibody; isolating and purifying the expressed anti-TIGIT monoclonal antibody. Using the above method, a crude anti-TIGIT monoclonal antibody can be obtained. The anti-TIGIT monoclonal antibody is then purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis, by a purification method, including TIGIT-based affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, molecular exclusion, purification on a protein A column, or any combination of these techniques.

[0110] In a sixth aspect, the present application provides a vaccine comprising the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect, and an optional immune adjuvant.

[0111] In general, the vaccine is prepared to be injectable, for example, a liquid solution or suspension, or a solid form suitable for resuspending in a liquid before injection. The preparation may also be emulsified. The active immunogenic component is usually mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are such as water, saline, glucose, glycerol, ethanol, etc. or a combination thereof. In addition, the vaccine may also contain a small amount of auxiliary materials as needed, such as a wetting agent or emulsifier, a pH buffer or an adjuvant that improves the effect of the vaccine.

[0112] The vaccine is given in a manner compatible with its dosage form, and the amount of administration is therapeutically effective and can produce immunity. The amount used depends on the subject being treated, including, for example, the ability of the individual immune system to produce an immune response, the route of administration, etc., and the specific amount is determined by the physician. For the first administration and booster injection, the applicable treatment regimen is also variable. In some embodiments, the vaccine is delivered intravenously, or directly delivered to the site of tumor or infection, or other traditional methods of vaccine administration.

[0113] In the seventh aspect, the present application provides the use of the antibody or its antigen-binding portion that specifically binds to TIGIT as described in the first aspect, the pharmaceutical composition as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the vaccine as described in the sixth aspect in the preparation of a drug for inhibiting Treg function, killing cells expressing TIGIT, inducing T cell-mediated responses, improving the function of effector T cells, improving the function of memory T cells, and / or effectively inhibiting tumor growth.

[0114] In the eighth aspect, the present application provides the use of the antibody or its antigen-binding portion that specifically binds to TIGIT as described in the first aspect, the pharmaceutical composition as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the vaccine as described in the sixth aspect in the preparation of a medicament for preventing and / or treating TIGIT-related diseases.

[0115] In the ninth aspect, the present application provides a method for preventing and / or treating TIGIT-related diseases, such as tumors, comprising administering to an individual in need the antibody or antigen-binding portion thereof that specifically binds to TIGIT as described in the first aspect, the pharmaceutical composition as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the vaccine as described in the sixth aspect.

[0116] In some embodiments, the method further comprises administering a second agent for preventing and / or treating a TIGIT-related disease such as a tumor, such as a chemotherapeutic agent and / or a PD-1 binding antagonist.

[0117] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. As used herein, "individual" and "subject" can be used interchangeably.

[0118] "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or alleviate (mitigate) the target pathological state or condition. Individuals in need of treatment include those who already have the condition, as well as those who will develop the condition or those for whom the condition is to be prevented. Therefore, the individual to be treated herein has been diagnosed as having the condition or is predisposed or susceptible to the condition.

[0119] The "therapeutically effective amount" used herein can be determined according to specific circumstances, and a person of ordinary skill in the art can easily determine the actual amount of medicine required, such as by the patient's weight, age and condition.

[0120] In an embodiment of the seventh or eighth aspect, the TIGIT-related disease is a tumor.

[0121] In some embodiments, the tumor described herein is a primary cancer or a metastatic cancer. In a specific embodiment, the tumor is selected from lung cancer, such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic cancer, such as leukemia, breast cancer, gastric cancer, esophageal cancer, B lymphocyte non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, head and neck cancer, such as head and neck squamous cell carcinoma, malignant glioma, kidney cancer, melanoma, prostate cancer, bone cancer, giant cell tumor of bone, pancreatic cancer, ovarian cancer, sarcoma, liver cancer, skin squamous cell carcinoma, thyroid cancer, cervical cancer, nasopharyngeal carcinoma, endometrial cancer, or metastatic cancer of the above tumors.

[0122] In embodiments of any aspect, the methods, uses and pharmaceutical compositions described herein may further comprise administering a second agent and / or treatment to the individual, for example as part of a combination therapy. Non-limiting examples of second agents and / or treatments can include radiation therapy, surgery, gemcitabine, cilastatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; vindesine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) that reduce cell proliferation. ) and VEGF-A inhibitors and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0123] In a ninth aspect, the present application provides a detection reagent or kit comprising the antibody or antigen-binding portion thereof described in the first aspect.

[0124] Antibodies or their antigen-binding portions as described herein can be combined with a detectable portion. Exemplary detectable portions include, but are not limited to, radioisotopes such as iodine 125, iodine-131, cesium-137, iridium 192, and cobalt 60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphatase, chemiluminescent agents such as luminols, etc. Those skilled in the art can select suitable detectable portions to be combined with antibodies or their antigen-binding portions of the present application as needed, thereby achieving different detection purposes.

[0125] Throughout the specification and claims, the words “comprises,” “comprising,” and “containing” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0126] It will be appreciated that features, characteristics, components or steps described in conjunction with a particular aspect, embodiment or example of the present application may be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0127] The present invention discloses antibodies that specifically bind to TIGIT in mammals (humans, primates, etc.), and the present invention provides applications of such proteins in treatment, screening, and detection, such as use in cancer treatment. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the scope of the present invention. Those skilled in the art can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the present invention.

[0128] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments.

[0129] Example

[0130] The following examples are used to illustrate the present application, but are not intended to limit the scope of the present application. Without departing from the spirit and substance of the present application, modifications or substitutions made to the methods, steps or conditions of the present application are within the scope of the present application.

[0131] Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0132] The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, for example, see Sambrook, J., Fritsch, EF. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0133] Example 1. Preparation of hybridomas producing anti-TIGIT antibodies

[0134] The preparation method is as follows:

[0135] 1. Take 6-8 week old Balb / c mice and inoculate TIGIT-His protein (100 μg / dose / mouse) intraperitoneally. TIGIT-His protein is constructed and expressed by the applicant. It contains the extracellular region M1-120P of the TIGIT sequence (mRNA: NM_173799.3) and a His tag at the C-terminus of the extracellular region. Its molecular weight is verified by SDS-PAGE to be 15 kDa. Immunization is performed once every 2 weeks, for a total of 3 times. Before fusion, mice are injected with TIGIT-His protein for 3 consecutive days.

[0136] 2. Two days before fusion, macrophages from the peritoneal cavity of common Kunming mice (KM) were taken as trophoblasts and inoculated into 96-well plates.

[0137] 3. Take lymphocytes from spleen and lymph nodes of immunized mice and fuse them with non-secretory myeloma SP2 / 0 cell line, add the fused cells to a 96-well plate pre-coated with a trophoblast layer, and perform HAT selection on the fused cells (Galfre and Milstein, Methods Enzymol 1981; 73: 3-46).

[0138] 4. Recover a group of hybridoma cells that all secrete anti-TIGIT specific antibodies. The initial screening is to determine the titer of anti-TIGIT antibodies secreted by the hybridoma using enzyme-linked immunosorbent assay (ELISA).

[0139] Example 2. Construction of human TIGIT (hTIGIT), monkey TIGIT (Cyno-TIGIT) and human PVR (hPVR) high-expressing cell lines

[0140] Human TIGIT (hTIGIT), monkey TIGIT (CynoTIGIT) and human PVR (hPVR) high-expressing cell lines were constructed using a stable cell line construction platform. The specific steps are as follows:

[0141] Construction of plasmids pEnter-hTIGIT-puromycin, pEnter-Cyno-TIGIT-puromycin and pEnter-hPVR-puromycin: hTIGIT cDNA (the amino acid sequence encoded by it is shown in SEQ ID NO: 17), Cyno-TIGIT cDNA (the amino acid sequence encoded by it is shown in SEQ ID NO: 18) and hPVR cDNA (the amino acid sequence encoded by it is shown in SEQ ID NO: 19) were used, respectively. NO: 19) was used as a template and primers were used for amplification. The amplified products were subjected to electrophoresis and gel recovery. The pEnter plasmid was digested with KpnI / HindIII and a large fragment of 7450 bp was recovered by gel. The three small fragments obtained above were respectively connected to the large fragment of the pEnter plasmid digested with KpnI / HindIII recovered by gel, and the positive clones were selected for sequencing. The correctly sequenced plasmids were named pEnter-hTIGIT-puromycin, pEnter-Cyno-TIGIT-puromycin and pEnter-hPVR-puromycin.

[0142] 293T cells (from Concord Cell Bank) were inoculated into three T25 culture flasks, with 2×10 cells in each culture flask. 6 . On the second day, the 293T cell culture medium was replaced with 4 mL Opti-MEM (Thermofisher Scientific Cat31985070). 5 μg of plasmids pEnter-hTIGIT-puromycin, pEnter-Cyno-TIGIT-puromycin and pEnter-hPVR-puromycin, which cloned human TIGIT (hTIGIT), monkey TIGIT (Cyno-TIGIT) and human PVR (hPVR), were added to Opti-MEM, with a final volume of 500 μL. Another 500 μL Opti-MEM was prepared, and 7 μL of transfection reagent PEI (3 μg / mL) was added thereto. The two were mixed and allowed to stand at room temperature for 20 minutes, and then added to the above-cultured 4 ml 293T cells. On the third day, the cell culture medium was replaced with 5 mL DMEM high-glucose medium. On the fourth day, 2 μg / mL puromycin was added for selection. After 2-3 days, a large number of cells died. Fresh culture medium was replaced until the cells grew stably, and then single clone screening, expansion culture and cryopreservation were performed.

[0143] The cell lines stably expressing the target gene constructed in this application are named 293T-hTIGIT cells, 293T-Cyno-TIGIT cells and 293T-hPVR cells. The protein sequences used are derived from publicly published databases, and the sequences of each protein are as follows:

[0144] SEQ ID NO: 17: Human TIGIT, NP_776160.2, T cell immunoreceptor with Ig and ITIM domain subtypes, [Homo sapiens]

[0145] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRI FLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

[0146] SEQ ID NO: 18: Cynomolgus monkey TIGIT, XP_014985302.2, T cell immunoreceptor with Ig and ITIM domain subtypes, [Macaca fascicularis, Crab-eating macaque, Cynomolgus monkey]

[0147] MRWCLFLIWAQGLRQAPLASGMMTGTIETTGNISAKKGGSVILQCHLSSTMAQVTQVNWEQHDHSLLAIRNAELGWHIYPAFKDRVAPGPGGLTLQSLTMNDTGEYFCTYHTYPDGTYRGR IFLEVLESSVAEHSARFQIPLLGAMAMMLVVICIAVIVVVVLARKKKSLRIHSVESGLQRKSTGQEEQIPSAPSPPGSCVQAEAAPAGLCGEQQGDDCAELHDYFNVLSYRSLGSCSFFTETG

[0148] SEQ ID NO: 19: Human PVR, NP_006496.4, polio receptor, [Homo sapiens]

[0149] MARAMAAAWPLLLVALLVLSWPPGTGDVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWIL VPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLIC NVTNALGARQAELTVQVKEGPPSEHSGMSRNAIIFLVLGILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTR

[0150] Example 3. Flow cytometric screening of antibodies secreted by hybridomas

[0151] Method 1: Screening of blocking clones

[0152] 293T-hPVR cells were collected, washed once with FACS buffer, and centrifuged at 2-5 × 10 5 cells / 50 μL / well were added to the flow tube, and then 50 μL of hybridoma supernatant and 500 ng / 10 μl of TIGIT-hFc protein (i.e., a fusion protein of the extracellular region of human TIGIT and human IgG1Fc, designed by the inventors of the present application) were added to each well, and the anti-TIGIT antibody of BMS was used as a control, followed by incubation for 0.5 hours.

[0153] 2 ml of FACS buffer was added to each well for washing twice, and APC goat anti-mouse IgG Fc secondary antibody (Biolegend, Cat405308) and anti-human IgG Fc secondary antibody (Biolegend, Cat409306) were added respectively. After incubation for 1 hour, the cells were detected by flow cytometry.

[0154] The results are shown in Table 1 and Figure 1 As shown. Six candidate monoclonal antibodies were obtained: 13H3, 16F5, 18A6, 18A12, 19F3 and 20D8. Blocking rate: Geom.Mean (TIGIT-hFc-样品) / Geom.Mean TIGIT-hFc *100%.

[0155] Table 1. Results of rescreening of anti-TIGIT blocking hybridoma clones

[0156] Hybridoma clone number Geom.Mean Blocking rate % TIGIT-hFc 11792 - 13H3 1579 88.6 16F5 1603 26.9 18A6 1709 41.0 18A12 2762 80.9 19F3 2667 86.6 20D8 2042 89.2 BMS 5372 54.3

[0157] Method 2: Screening of high-affinity clones of cynomolgus monkey TIGIT

[0158] 293T-Cyno-TIGIT cells were collected, washed once with FACS buffer, and centrifuged at 2-5 × 10 5 10 cells / 50 μL / well were added to the flow tube, and then 50 μL of the above 6 hybridoma supernatants were added to each well, followed by incubation for 0.5 hours.

[0159] 2 ml of FACS buffer was added to each well and washed twice. APC goat anti-mouse IgG Fc secondary antibody (Biolegend, Cat405308) was added respectively, and the cells were incubated for 1 hour before detection by flow cytometry.

[0160] The results are shown in Table 2 and Figure 2 As shown:

[0161] Table 2. Screening of high affinity hybridoma clones against cynomolgus monkey TIGIT

[0162] Hybridoma clone number Geom.Mean 293T-Cyno-TIGIT 826 APC goat anti-mouse IgG Fc 1918 13H3 2470 16F5 2049 18A6 1911 18A12 19972 19F3 5070 20D8 1966

[0163] Through the above two methods, hybridoma clone 18A12 was obtained in this example, and the antibody in its supernatant can effectively block the binding of human TIGIT and PVR, and can bind to TIGIT of cynomolgus monkey with high affinity. Hybridoma clone 18A12 was sequenced to obtain heavy chain CDRs shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4, and light chain CDRs shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7)

[0164] Example 4. Binding activity of anti-TIGIT chimeric antibody and 293T-hTIGIT cells (FACS detection)

[0165] Preparation of anti-TIGIT chimeric antibodies

[0166] The cDNA of 18A12 hybridoma cells was obtained and amplified by PCR and sequenced using the antibody primers stored in this laboratory, and finally the gene sequences encoding VH1 and VL1 of the mouse anti-TIGIT antibody were obtained. The nucleic acid sequences encoding VH1 and VL1 of the anti-TIGIT antibody were constructed into plasmids pUC57 TIGIT 18A12-VH1 and pUC57 TIGIT 18A12-VL1 (General Biosystems (Anhui) Co., Ltd.) by total synthesis. The anti-TIGIT VH1 and TIGIT VL1 fragments were amplified using the Gold Mix PCR Kit (TSINGKE) according to the instructions of the kit, using pUC57 TIGIT 18A12-VH1 and pUC57 TIGIT 18A12-VL1 as templates, respectively. At the same time, the vector plasmids pQKX1 and pQKX2 were digested with the restriction endonuclease SapI (NEB, R0569S). The resulting PCR amplification products and the digested vectors were recombined and ligated using the BM Seamless Cloning Kit (Bomed) according to the instructions of the kit to obtain the heavy chain expression vector pQK TIGIT 18A12-H1 and the light chain expression vector pQK TIGIT18A12-L1.

[0167] 293Fv cells (Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) were diluted to 1.5×10 6 cells / mL, the final volume was 200 ml, and the cells were cultured on a shaker at 37°C for 24 h. Plasmids pQKTIGIT 18A12-H1 and pQK TIGIT 18A12-L1 were diluted with fresh culture medium at 10% of the transfection volume to a concentration of 1 μg / mL calculated based on the volume of transfected cells. To the diluted plasmid, 200 μl of 3 mg / mL PEI was added at 1 / 1000 of the cell volume, and the mixture was immediately vortexed for 10 seconds and placed at room temperature for 15 minutes. The plasmid / PEI mixture was added dropwise to the cell culture medium, and the culture bottle was gently shaken while adding dropwise, and placed in a shaker for culture. After 7 days, the culture supernatant was collected, and after filtering with a 0.4 μm filter membrane, the antibody was captured from the culture supernatant using a Mab sure Lx 5ml purification column. The flow rate was set to 3 ml / min, and the purification column was equilibrated with 5 column volumes of 20 mM PB + 150 mM Nacl, pH 7.4 equilibrium solution. After the column equilibrium was stable, the sample was loaded. After the loading was completed, 20 mM PB + 150 mM Nacl, pH 7.4 was selected for elution. After the elution was completed, 50 mM citric acid pH 3.0 eluent was used for elution, and the antibody eluted by neutralization with 1 M Tris-Hcl, pH 9.0 was collected (the amino acid sequences of the heavy chain and light chain were as shown in SEQ ID NO:10 and SEQ ID NO:11).

[0168] Collect 293T-hTIGIT cells from a T75 cell culture flask, wash once with FACS buffer, resuspend and count with FACS buffer, and adjust the cell concentration to 2 × 10 6 ; Dilute antibodies (including BMS TIGIT Ab and anti-TIGIT chimeric antibody, with BMS TIGIT Ab as control) in a gradient dilution. Take 10 μL of each diluted antibody and add 100 μL of the cell sample after adjusting the concentration, mix well, and incubate at room temperature for 1 hour.

[0169] Each well was washed twice with 2 ml of FACS buffer, and APC anti-human IgG Fc secondary antibody (Biolegend, Cat409306) was added, incubated for 1 hour, and detected by flow cytometry. The results are shown in Table 3 and Figure 3 . Figure 3 The results showed that both anti-TIGIT chimeric antibody and control antibody BMS TIGIT Ab specifically bound to 293T-hTIGIT.

[0170] Table 3. Binding activity of anti-TIGIT chimeric antibodies to 293T-hTIGIT

[0171] Grouping Geom.Mean APC anti-human IgG Fc 405 Chimeric antibody 1.7 μg 12194 Chimeric antibody 1.0 μg 10331 Chimeric antibody 0.5 μg 8786

[0172] Example 5. In vivo efficacy experiment of anti-TIGIT chimeric antibody in animals

[0173] A mouse colon cancer model was established subcutaneously in B-hTIGIT mice (Southern model) using mouse colon cancer cell line MC38 cells. Then, anti-TIGIT chimeric antibodies and PD-1 antibodies (BE0033-2) were administered to investigate the inhibitory effects of single and combined drugs on mouse tumors.

[0174] Materials and Methods

[0175] 5 × 10 were injected subcutaneously into the dorsal aspect of the right forelimb 5 MC38 cells were used to establish a mouse colon cancer model. When the tumor volume was 100-150 mm 3 At the same time, the mice were divided into groups according to the tumor volume: control: normal saline; PD-1 group: 1 mg / kg; TIGIT group: 10 mg / kg; PD-1 and TIGIT combined group: 1+10 mg / kg. Then the monoclonal antibody was injected intraperitoneally twice a week for a total of 6 times. After administration, the changes in the physical signs of the mice were closely observed, and the tumor size and body weight were measured and recorded before each administration.

[0176] Results Figure 4In terms of efficacy, the tumor inhibition efficiency of the combined group was significantly better than that of the control group and the single drug group. At the end of the experiment, the tumors were dissected and the tumor size was analyzed. There was no significant difference between the groups. The relative tumor inhibition rate TGI is shown in Table 4. In summary, the combination of PD-1 and TIGIT can effectively inhibit tumor growth.

[0177] Table 4. Tumor inhibition relative rate (TGI) of anti-TIGIT chimeric antibodies

[0178]

[0179]

[0180] Note: D3 means the 3rd day after administration, D7 means the 7th day after administration, and so on.

[0181] Example 6. Binding activity of anti-TIGIT humanized antibodies and 293T-hTIGIT cells, 293T-Cyno-TIGIT cells and 293T-hPVR (FACS detection)

[0182] Preparation of humanized anti-TIGIT antibody

[0183] The nucleotide sequences encoding VH2 and VL2 of the anti-TIGIT humanized antibody were designed by the inventors of the present application. The anti-TIGIT humanized antibody VH2 and VL2 nucleotide sequences were constructed into plasmids pUC57 TIGIT VH2 and pUC57TIGIT VL2 (General Biosystems (Anhui) Co., Ltd.) by total synthesis. pUC57 TIGIT VH2 and pUC57 TIGIT VL2 were used as templates, respectively, and the anti-TIGIT VH2 and TIGIT VL2 fragments were amplified using the Gold Mix PCR Kit (TSINGKE) according to the instructions of the kit. At the same time, the vector plasmids pQKX1 and pQKX2 were digested with the restriction endonuclease SapI (NEB, R0569S), and the resulting PCR amplification products and digested vectors were recombined and ligated using the BM seamless cloning kit (Bomed) according to the instructions of the kit to obtain the heavy chain expression vector pQK 18A12 H2 and the light chain expression vector pQK 18A12 L2.

[0184] Dilute 293Fv cells to 1.5 × 10 6cells / mL, the final volume was 200 ml, and cultured at 37°C in a shaking incubator for 24 h. Plasmids pQK18A12 H2 and pQK 18A12 L2 were diluted with fresh culture medium at 10% of the transfection volume to a concentration of 1 μg / mL calculated based on the volume of transfected cells. Add 200 μl of 3 mg / mL PEI to the diluted plasmid at 1 / 1000 of the cell volume, vortex immediately for 10 seconds, leave at room temperature for 15 minutes, add the plasmid / PEI mixture dropwise to the cell culture medium, gently shake the culture bottle while adding, and culture on a shaker. Collect the culture supernatant after 7 days, filter with a 0.4 μm filter membrane, and use Mab sure Lx5ml purification column to capture antibodies from the culture supernatant. The flow rate is set to 3 ml / min, and the purification column is equilibrated with 5 column volumes of 20 mM PB + 150 mM Nacl, PH7.4 equilibrium solution. Load the sample after the column equilibrium is stable. After loading, select 20 mM PB + 150 mM Nacl, PH7.4 for elution. After elution, use 50 mM citric acid PH3.0 eluent for elution, and collect the eluted antibodies with 1 M Tris-Hcl, PH9.0. A total of 6 anti-TIGIT humanized antibodies were prepared and numbered #1 to #6. The heavy chain variable regions and light chain variable regions of anti-TIGIT humanized antibodies #1 to #6 are shown in Table 5.

[0185] Table 5. Heavy chain variable regions and light chain variable regions of anti-TIGIT humanized antibodies #1-#6

[0186]

[0187] Binding activity of anti-TIGIT humanized antibody and 293T-hTIGIT cells

[0188] Collect 293T-hTIGIT cells in T75 cell culture flask, wash once with FACS buffer, resuspend and count with FACS buffer, and adjust the cell concentration to 2×10 6 ; Dilute antibodies (including BMS TIGIT Ab and six anti-TIGIT humanized antibodies, of which BMS TIGIT Ab was used as a control). Take 10 μL of each diluted antibody and add 100 μL of the cell sample after adjusting the concentration, mix well, and incubate at room temperature for 1 hour.

[0189] Each well was washed twice with 2 ml of FACS buffer, and APC goat anti-human IgG Fc secondary antibody (Biolegend, Cat409306) was added, incubated for 1 hour, and detected by flow cytometry. The results are shown in Figure 5 . Figure 5 The results showed that both anti-TIGIT humanized antibody and control antibody BMS TIGIT Ab specifically bound to 293T-hTIGIT cells.

[0190] Binding activity of anti-TIGIT humanized antibody and 293T-Cyno-TIGIT cells

[0191] Collect 293T-Cyno-TIGIT cells in T75 cell culture flask, wash once with FACS buffer, resuspend and count with FACS buffer, and adjust the cell concentration to 2×10 6 ; Dilute antibodies (including BMS TIGIT Ab and six anti-TIGIT humanized antibodies, of which BMS TIGIT Ab was used as a control). Take 10 μL of each diluted antibody and add 100 μL of the cell sample after adjusting the concentration, mix well, and incubate at room temperature for 1 hour.

[0192] Each well was washed twice with 2 ml of FACS buffer, and APC goat anti-human IgG Fc secondary antibody (Biolegend, Cat409306) was added, incubated for 1 hour, and detected by flow cytometry. The results are shown in Figure 6 . Figure 6 The results showed that the affinity of the control antibody BMS TIGITAb to 293T-Cyno-TIGIT cells was much stronger than that of the TIGIT humanized antibody.

[0193] Binding activity of anti-TIGIT humanized antibody and 293T-hPVR cells

[0194] Collect 293T-hPVR cells in T75 cell culture flask, wash once with FACS buffer, resuspend and count with FACS buffer, and adjust the cell concentration to 2×10 6 ; Dilute antibodies (including BMS TIGIT Ab and six anti-TIGIT humanized antibodies, of which BMS TIGIT Ab was used as a control). Take 10 μL of each diluted antibody and 500 ng of TIGIT-hFc and add them to 100 μL of the cell sample after adjusting the concentration, mix well, and incubate at room temperature for 1 hour.

[0195] Each well was washed twice with 2 ml of FACS buffer, and APC goat anti-human IgG Fc secondary antibody (Biolegend, Cat409306) was added, incubated for 1 hour, and detected by flow cytometry. The results are shown in Figure 7 . Figure 7 The results showed that the control antibody BMS TIGITAb and the TIGIT humanized antibody both had strong blocking ability.

[0196] Example 7. In vivo efficacy experiment of humanized anti-TIGIT antibodies in animals

[0197] MC38 Model

[0198] A mouse colon cancer model was established subcutaneously in B-hTIGIT mice (Southern model) using the mouse colon cancer cell line MC38. Then, #2 anti-TIGIT humanized antibody and anti-PD-1 antibody (BE0033-2) were administered to investigate the inhibitory effects of single and combined drugs on mouse tumors.

[0199] Materials and Methods

[0200] MC38 cells were used as target cells, 5e 5 The amount of cells / mouse was inoculated subcutaneously in the right forelimb of B-hTIGIT mice. When the tumor grew to 80-100mm 3 The mice were measured, divided into groups, and given medication. The mice were then measured and given medication twice a week, and the weight of the mice and the length and width of the tumor were recorded. The tumor volume was calculated as: V = L*W2 / 2. When the tumor grew to the ethical endpoint (2000mm 3 ), the mice were killed by CO2, and the tumor weight, spleen weight and body weight of the mice were measured and photographed. The ratio of tumor weight to body weight was calculated.

[0201] Tumor volume changes Figure 8 As shown in A, the tumor inhibition effect of the combined drug is fully demonstrated. Compared with the control group, the antibody group has a certain tumor inhibition effect and there is a significant statistical difference between the two groups (p < 0.05). As can be seen from Table 6, the relative tumor inhibition rate of the combined drug group is the highest, reaching 80%. Although the weight of the mice in the drug group is lighter than that of the control group ( Figure 8 B), but the difference did not increase further, indicating that the drug had little effect on the body weight of mice and there was no significant statistical difference compared with the control group (p>0.05).

[0202] In addition, at the end of the experiment, the mice were dissected and the weight percentage of the tumor tissue and spleen tissue of the mice was measured. Compared with the control group, the weight percentage of the tumor in the drug-treated group was reduced, with a significant statistical difference (p < 0.05); compared with the control group, the weight percentage of the spleen in the drug-treated group was reduced, with a significant statistical difference (p < 0.05). Combined with the tumor weight index, it was inferred that it was positively correlated with the tumor size.

[0203] Table 6. Relative tumor inhibition rate (TGI%) of the combined group of tumor inhibition experiment

[0204]

[0205] Note: D3 means the third day after administration, D7 means the seventh day after administration, and so on. PD1+TIGIT means the combined administration of anti-PD-1 antibody + anti-TIGIT humanized antibody.

[0206] CT26 Model

[0207] A mouse colon cancer model was established subcutaneously in BALB / C-hTIGIT mice (Jicui Yaokang) using the mouse colon cancer cell line CT26. Then, #2 anti-TIGIT humanized antibody and anti-PD-1 antibody (BE0033-2) were administered to investigate the inhibitory effects of single and combined drugs on mouse tumors.

[0208] Using CT26 cells as target cells, 5e 5 The amount of cells / mouse was inoculated subcutaneously in the right forelimb of B-hTIGIT mice. When the tumor grew to 80-100mm 3 The mice were measured, divided into groups, and given medication. Then, the mice were measured and given medication twice a week, and the weight of the mice and the length and width of the tumor were recorded. The tumor volume was calculated as: V = L*W2 / 2. When the tumor grew to the ethical endpoint (2000mm 3 ), the mice were killed by CO2, and the tumor weight, spleen weight and body weight of the mice were measured and photographed. The ratio of tumor weight to body weight was calculated.

[0209] The results of the tumor inhibition experiment of anti-TIGIT humanized antibody in CT26 tumor model are as follows Fig. 9 As shown in A, the tumor inhibition effect of the PD1+TIGIT combined administration group was better than that of the PD-1 and TIGIT single drug groups, and there were significant statistical differences between the groups (p < 0.05). There was no significant difference in the weight of mice in each group, and there was no significant difference (p > 0.05).

[0210] At the end of the experiment, the mice were dissected and the weight proportions of tumor tissue and spleen tissue were measured. Fig.11 Compared with the control group, the weight percentage of the tumor in the drug-treated group decreased, with no significant statistical difference (p>0.05); compared with the control group, the weight percentage of the spleen in the drug-treated group was significantly different, with statistical difference (p<0.05). The results of the relative tumor inhibition rate TGI (%) are shown in Table 7. As the drug-treated time progresses, the TGI of the drug-treated group gradually increases.

[0211] Table 7. Relative tumor inhibition rate (TGI%) of the combined group of tumor inhibition experiment

[0212]

[0213] Note: D3 means the third day after administration, D7 means the seventh day after administration, and so on. PD1 means anti-PD-1 antibody alone; TIGIT means anti-TIGIT humanized antibody alone; PD1+TIGIT means anti-PD-1 antibody + anti-TIGIT humanized antibody combined administration.

[0214] It is understood that although the present application describes the inventions involved in the above specific forms, these inventions are not limited to the specific contents described in these specific forms. It is obvious to those skilled in the art that, without departing from the spirit of the invention described in the present application, various equivalent changes may be made to the technical features included in the inventions involved therein, and these changes should all fall within the scope of the invention. Sequence Listing <110> Beijing Immune Ark Pharmaceutical Technology Co., Ltd. <120> Anti-TIGIT antibodies and their applications <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <400> 1 Gly Phe Thr Phe Ser Asn Tyr Trp 1 5 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <400> 2 Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <400> 3 Ala Arg Leu Tyr Tyr Gly Asn Tyr Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Thr Arg Leu Tyr Tyr Gly Asn Tyr Phe Asp Tyr 1 5 10 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <400> 5 Glu Asn Thr Tyr Ser Tyr 1 5 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <400> 6 Asn Ala Lys 1 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Gln His His Tyr Ala Phe Ser Tyr Thr 1 5 <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <400> 8 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Thr Arg Leu Tyr Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Thr Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Thr Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Ala Phe Ser Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 100 105 <210> 10 <211> 450 <212> PRT <213> Artificial Sequence <400> 10 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Thr Arg Leu Tyr Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 11 <211> 214 <212> PRT <213> Artificial Sequence <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Thr Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Thr Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Ala Phe Ser Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Tyr Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <400> 13 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Tyr Tyr Gly Asn Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Thr Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ala Phe Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Thr Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ala Phe Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Thr Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ala Phe Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 244 <212> PRT <213> Artificial Sequence <400> 17 Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35 40 45 Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55 60 Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser 65 70 75 80 Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln 85 90 95 Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr 100 105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu 115 120 125 Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly 130 135 140 Ala Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val 145 150 155 160 Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu 165 170 175 Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser 180 185 190 Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala 195 200 205 Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp 210 215 220 Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe 225 230 235 240 Thr Glu Thr Gly <210> 18 <211> 245 <212> PRT <213> Artificial Sequence <400> 18 Met Arg Trp Cys Leu Phe Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala Lys Lys Gly Gly Ser Val Ile Leu Gln Cys His Leu Ser 35 40 45 Ser Thr Met Ala Gln Val Thr Gln Val Asn Trp Glu Gln His Asp His 50 55 60 Ser Leu Leu Ala Ile Arg Asn Ala Glu Leu Gly Trp His Ile Tyr Pro 65 70 75 80 Ala Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu 85 90 95 Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His 100 105 110 Thr Tyr Pro Asp Gly Thr Tyr Arg Gly Arg Ile Phe Leu Glu Val Leu 115 120 125 Glu Ser Ser Val Ala Glu His Ser Ala Arg Phe Gln Ile Pro Leu Leu 130 135 140 Gly Ala Met Ala Met Met Leu Val Val Ile Cys Ile Ala Val Ile Val 145 150 155 160 Val Val Val Leu Ala Arg Lys Lys Lys Ser Leu Arg Ile His Ser Val 165 170 175 Glu Ser Gly Leu Gln Arg Lys Ser Thr Gly Gln Glu Glu Gln Ile Pro 180 185 190 Ser Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro 195 200 205 Ala Gly Leu Cys Gly Glu Gln Gln Gly Asp Asp Cys Ala Glu Leu His 210 215 220 Asp Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Ser Cys Ser Phe 225 230 235 240 Phe Thr Glu Thr Gly 245 <210> 19 <211> 417 <212> PRT <213> Artificial Sequence <400> 19 Met Ala Arg Ala Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu 1 5 10 15 Leu Val Leu Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln 20 25 30 Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro 35 40 45 Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu 50 55 60 Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln 65 70 75 80 Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala 85 90 95 Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly 100 105 110 Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe 115 120 125 Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys 130 135 140 Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro 145 150 155 160 Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln 165 170 175 Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val 180 185 190 Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu 195 200 205 Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu 210 215 220 His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val 225 230 235 240 Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr 245 250 255 Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro 260 265 270 Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro 275 280 285 Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys 290 295 300 Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala 305 310 315 320 Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu 325 330 335 His Ser Gly Met Ser Arg Asn Ala Ile Ile Phe Leu Val Leu Gly Ile 340 345 350 Leu Val Phe Leu Ile Leu Leu Gly Ile Gly Ile Tyr Phe Tyr Trp Ser 355 360 365 Lys Cys Ser Arg Glu Val Leu Trp His Cys His Leu Cys Pro Ser Ser 370 375 380 Thr Glu His Ala Ser Ala Ser Ala Asn Gly His Val Ser Tyr Ser Ala 385 390 395 400 Val Ser Arg Glu Asn Ser Ser Ser Gln Asp Pro Gln Thr Glu Gly Thr 405 410 415 Arg

Claims

1. An antibody or an antigen-binding portion thereof that specifically binds to TIGIT, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is GFTFSNYW (SEQ ID NO: 1), the HCDR2 sequence is IRLKSNNYAT (SEQ ID NO: 2), the HCDR3 sequence is ARLYYGNYFDY (SEQ ID NO: 3) or TRLYYGNYFDY (SEQ ID NO: 4), and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1 sequence is ENTYSY (SEQ ID NO: 5), the LCDR2 sequence is NAK (SEQ ID NO: 6), and the LCDR3 sequence is QHHYAFSYT (SEQ ID NO: 7).

2. The antibody or antigen-binding portion thereof according to claim 1, wherein the antigen-binding portion is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.

3. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the antibody is a murine antibody, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

4. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the antibody is a chimeric antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 10, and a light chain having an amino acid sequence as shown in SEQ ID NO:

11.

5. The antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody is a humanized antibody, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 12 or SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

6. The antibody or antigen-binding portion thereof according to claim 1 or 2, which specifically binds to primate TIGIT.

7. The antibody or antigen-binding portion thereof of claim 6, wherein the primate TIGIT is selected from human TIGIT or monkey TIGIT.

8. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the antibody or antigen-binding portion thereof is 1×10 -10 Up to 1×10 -9 The KD of M specifically binds to the TIGIT molecule.

9. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the antibody has a PVR inhibitory function and stimulates the activation and proliferation of T cells.

10. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier, 11. The pharmaceutical composition of claim 10, further comprising a chemotherapeutic agent and / or a PD-1 binding antagonist.

12. A nucleic acid molecule encoding the antibody or antigen binding portion thereof of any one of claims 1-9.

13. An expression vector comprising the nucleic acid molecule of claim 12.

14. A host cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13.

15. Use of the antibody or antigen-binding portion thereof according to any one of claims 1 to 9, the pharmaceutical composition according to claim 10, or the host cell according to claim 14 in the preparation of a medicament for treating a TIGIT-related disease, wherein the disease is a tumor, and the tumor is selected from one or more of the following: colon cancer, lung cancer, cervical cancer, esophageal cancer, gastric cancer, and metastatic tumors thereof.

16. A detection reagent or kit comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 9.

Images