High affinity tigit antibodies and uses thereof

Abstract

The application provides an antibody or an antigen binding fragment thereof and application thereof. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region CDR1, CDR2 and CDR3 sequence as shown in SEQ ID NO: 1, 2 and 3 or an amino acid sequence with at least 80% identity with SEQ ID NO: 1, 2 and 3; and a light chain variable region CDR1, CDR2 and CDR3 sequence as shown in SEQ ID NO: 4, 5 and 6 or an amino acid sequence with at least 80% identity with SEQ ID NO: 4, 5 and 6. The antibody or the antigen binding fragment thereof can bind to TIGIT, block the binding of TIGIT to a ligand CD155, release TIGIT-mediated immunosuppression, activate an immune cell anti-cancer mechanism, and then inhibit the growth and proliferation of tumors.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to a high-affinity TIGIT antibody and its applications, and more specifically to an antibody or its antigen-binding fragment, nucleic acid molecules, expression vectors, methods for preparing antibodies or their antigen-binding fragments, recombinant cells, compositions and their uses, and pharmaceuticals and their uses. Background Technology

[0002] Malignant tumors are a serious threat to human life and health. Current treatments for malignant tumors, including surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, immune checkpoint therapy, cell and gene therapy, can only play a limited role in a portion of malignant tumor patients. Malignant tumors remain a difficult problem troubling human life and health.

[0003] In recent years, immunotherapy has made remarkable progress in the field of cancer treatment, especially immune checkpoint blockade therapy represented by anti-CTLA-4 and anti-PD-1 or PD-L1 antibodies. By blocking the binding of inhibitory receptors on the surface of T cells to their ligands, these therapies block the transmission of inhibitory signals, correct immunosuppression mediated by the immunosuppressive microenvironment, and restore the anti-tumor ability of T cells in the tumor microenvironment. They have achieved high response rates in the treatment of various metastatic advanced malignancies (including metastatic melanoma, non-small cell lung cancer, and renal cell carcinoma). However, it cannot be ignored that even the PD-1 / L1 therapy, which currently has the broadest approved indications, still has an overall response rate of only 30%, meaning that many more patients cannot benefit from it.

[0004] However, not all patients with malignant tumors respond to PD-1, PD-L1, or CTLA-4 blockade therapy. Only 10%-30% of patients show a long-lasting response to PD-1 or PD-L1 antibody therapy, with the majority lacking a response. On the one hand, the significant heterogeneity in the expression of immune checkpoint ligands in tumors and tumor-infiltrating lymphocytes means that a single type of immune checkpoint therapy cannot be applied to all patients, and most do not benefit from it. On the other hand, some patients who have received immune checkpoint therapy experience tumor recurrence and develop tolerance to that therapy, rendering continued administration ineffective—a phenomenon known as secondary resistance. For these two reasons, it is necessary to develop immune checkpoint antibodies targeting more specific targets.

[0005] T-cell immunoglobulins and the ITIM (Immunoreceptor tyrosine-based inhibitory motif) domain protein (TIGIT) serve as important immune checkpoints, primarily expressed on the surface of natural killer (NK) cells, activated CD8+ and CD4+ T cells, regulatory T cells (Tregs), and follicular helper T cells (Tfh). TIGIT-recognized ligands CD155 and CD112 are mainly expressed on monocytes, macrophages, dendritic cells (DCs), T cells, B cells, and many non-hematopoietic cells (including tumor cells of various histological types). TIGIT binds to its ligand PVR (poliovirus receptor, CD155) with significantly higher affinity than its competitive receptors CD226 and CD96. TIGIT binding to its ligands transmits inhibitory signals via the intracellular ITIM motif, thereby suppressing the function of T cells and NK cells.

[0006] Studies have shown that TIGIT expression is associated with the development of various malignant tumors, such as non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, colorectal cancer, glioblastoma, gastric cancer, liver cancer, multiple myeloma, acute myeloid leukemia, and follicular lymphoma. It has been reported that tumor-infiltrating T cells in lung cancer patients highly express TIGIT molecules, and its expression is significantly positively correlated with PD-1 expression. In colorectal cancer patients, TIGIT expression on the surface of tumor-infiltrating NK cells is significantly higher than that on peripheral blood NK cells. In various mouse tumor models, blocking TIGIT with antibodies can inhibit tumor growth and metastasis, and blocking both TIGIT and PD-1 can effectively treat tumors. Therefore, the development of therapeutic TIGIT antibodies is particularly necessary. Summary of the Invention

[0007] The present invention aims to at least partially solve one of the technical problems in the related art.

[0008] In order to inhibit tumor growth and metastasis and the resulting secondary drug resistance, this invention provides a high-affinity TIGIT antibody and its application in the treatment of malignant tumors.

[0009] The antibody provided by this invention relieves TIGIT-mediated immunosuppression and promotes the anti-cancer function of immune cells by blocking the binding of TIGIT to its ligand CD155.

[0010] Therefore, in order to achieve the above objectives, in a first aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0011] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO: 1, 2, and 3 or amino acid sequences having at least 80% identity with SEQ ID NO: 1, 2, and 3; and

[0012] The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO: 4, 5, and 6, or amino acid sequences having at least 80% identity with 4, 5, and 6, respectively.

[0013] According to embodiments of the present invention, the antibody or its antigen-binding fragment can bind to TIGIT, blocking the binding of TIGIT to the ligand CD155, relieving TIGIT-mediated immunosuppression, activating the anti-cancer mechanism of immune cells, and thereby inhibiting tumor growth and proliferation. Furthermore, the antibody or its antigen-binding fragment has high specificity, which can avoid the occurrence of secondary drug resistance.

[0014] According to embodiments of the present invention, the antibody or its antigen-binding fragment may further include at least one of the following additional technical features:

[0015] According to embodiments of the present invention, the antibody or its antigen-binding fragment further comprises:

[0016] The heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, the heavy chain variable region CDR2 shown in SEQ ID NO: 2, the heavy chain variable region CDR3 shown in SEQ ID NO: 3, the light chain variable region CDR1 shown in SEQ ID NO: 4, the light chain variable region CDR2 shown in SEQ ID NO: 5, and the light chain variable region CDR3 shown in SEQ ID NO: 6.

[0017] GSSITSDYA (SEQ ID NO: 1),

[0018] ITYSGRT (SEQ ID NO: 2),

[0019] ARWGLLRRYFDY(SEQ ID NO:3),

[0020] QDVFNQ(SEQ ID NO: 4),

[0021] SASFRYT (SEQ ID NO: 5),

[0022] QQHYSTPLT (SEQ ID NO: 6).

[0023] According to an embodiment of the present invention, the antibody or its antigen-binding fragment specifically recognizes TIGIT.

[0024] According to an embodiment of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region being as shown in SEQ ID NO: 8.

[0025] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGR TTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTV SS (SEQ ID NO: 7),

[0026] DIVMTQSPSSMSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTG VPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELK (SEQ ID NO: 8).

[0027] According to an embodiment of the present invention, the antibody is a humanized antibody.

[0028] According to an embodiment of the present invention, the antibody or its antigen-binding fragment contains a heavy chain framework region sequence and a light chain framework region sequence, wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0029] According to embodiments of the present invention, the antibody or its antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, a mouse antibody, or a mutant thereof.

[0030] According to embodiments of the present invention, both the light chain constant region and the heavy chain constant region are derived from murine IgG1 antibody, IgG2a antibody or its mutant, or human IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody or its mutant. According to embodiments of the present invention, the antibody or its antigen-binding fragment is at least one of single-chain antibody, multimeric antibody, CDR transplantation antibody, Fab antibody, and Fv antibody.

[0031] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0032] The antibodies encoded by nucleic acid molecules or their antigen-binding fragments according to embodiments of the present invention have stronger specificity, longer half-life and higher potency, enabling effective treatment or prevention of malignant tumors with lower drug doses, and with lower toxicity and side effects and higher safety.

[0033] In a third aspect, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect of the present invention.

[0034] The expression vector proposed in this invention can efficiently express the aforementioned antibody or its antigen-binding fragment in suitable recipient cells. The antibody or its antigen-binding fragment designed in this invention has stronger specificity and higher safety.

[0035] In a fourth aspect, the present invention provides a method for preparing the above-described antibody or its antigen-binding fragment. According to an embodiment of the present invention, the method includes:

[0036] The expression vector described in the third aspect of the present invention is introduced into cells;

[0037] The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the antibody or its antigen-binding fragment.

[0038] The inventors have discovered that antibodies or antigen-binding fragments prepared according to embodiments of the present invention can be cultured more efficiently to produce high-purity antibodies or antigen-binding fragments, and the operation steps are simple and the cost is low.

[0039] According to an embodiment of the present invention, the cell is a eukaryotic cell.

[0040] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell expresses the antibody or its antigen-binding fragment described in the first aspect of the present invention, and carries the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention.

[0041] According to some specific embodiments of the present invention, the recombinant cells can efficiently and extensively express antibodies or their antigen-binding fragments under suitable conditions. The antibodies or their antigen-binding fragments have stronger specificity, longer half-life and higher potency, enabling the delivery of antibody drugs to target cells with a smaller drug dose, thereby achieving effective treatment of diseases or prevention of cancer. They have low toxicity and higher safety.

[0042] In a sixth aspect, the present invention provides a composition. According to embodiments of the present invention, the composition comprises: an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, an expression vector as described in the third aspect of the present invention, or a recombinant cell as described in the fifth aspect of the present invention.

[0043] According to embodiments of the present invention, the composition of the present invention can achieve the same therapeutic effect as antibodies or their antigen-binding fragments, nucleic acid molecules, expression vectors, and recombinant cells when introduced into a living organism.

[0044] In a seventh aspect, the invention provides for the use of a composition in the preparation of a medicament. According to embodiments of the invention, the medicament is used to treat or prevent malignant tumors.

[0045] According to embodiments of the present invention, the antibody or its antigen-binding fragment can be used to prepare a drug, the prepared drug having the same efficacy as the antibody or its antigen-binding fragment, and can be used to treat or prevent diseases such as malignant tumors.

[0046] According to embodiments of the present invention, the malignant tumor includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0047] In an eighth aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises: an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, an expression vector as described in the third aspect of the present invention, recombinant cells as described in the fifth aspect of the present invention, or a composition as described in the sixth aspect of the present invention, the medicament being used to treat malignant tumors.

[0048] The inventors have discovered that drugs containing antibodies or their antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, or compositions have superior efficacy in the treatment and prognosis of malignant tumors.

[0049] According to embodiments of the present invention, the malignant tumor includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0050] In a ninth aspect of the invention, the invention provides for the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit. According to embodiments of the invention, the kit is used for the detection of TIGIT.

[0051] According to embodiments of the present invention, the kit provides more efficient and accurate detection of TIGIT, saving time and exploration costs for clinical treatment.

[0052] In a tenth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0053] The inventors discovered that kits prepared based on antibodies or their antigen-binding fragments can be applied to scientific research, saving time and costs associated with analyzing malignant tumors. Attached Figure Description

[0054] Figure 1 This is an ELISA result diagram of the binding of TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) to human and monkey TIGIT according to Example 3 of the present invention;

[0055] Figure 2 This is an ELISA result diagram of the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) blocking the binding of TIGIT to CD155 according to Example 4 of the present invention.

[0056] Figure 3 This is a diagram showing the results of the binding of the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) to 293T-TIGIT cells according to Example 5 of the present invention.

[0057] Figure 4 This is a diagram showing the results of blocking the binding of CD155-mIgG2a antibody to 293T-TIGIT cells by the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) according to Example 6 of the present invention.

[0058] Figure 5 This is a diagram showing the results of promoting IL-2 secretion by Jurkat T cells using the TIGIT antibody H401LV2-hlgG1LALA according to Example 7 of the present invention.

[0059] Figure 6 This is a diagram showing the results of the TIGIT antibody H401LV2-mlgG2a binding to CHO-K1-TIGIT cells according to Example 8 of the present invention.

[0060] Figure 7 This is a diagram showing the results of blocking CD155 binding to CHO-K1-TIGIT cells with the TIGIT antibody H401LV2-mlgG2a according to Example 9 of the present invention.

[0061] Figure 8 This is a graph showing the efficacy of the TIGIT antibody H401LV2-mIgG2a in the colorectal cancer MC38 model according to Example 10 of the present invention.

[0062] Figure 9 This is a graph showing the efficacy results of H401LV2-mIgG2aD265A in a lung cancer LLC model according to Example 10 of the present invention. Detailed Implementation

[0063] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0064] In the process of describing this invention, the terms used herein have been explained and described. These explanations and descriptions are only for the purpose of facilitating the understanding of the solution and should not be regarded as a limitation on the protection of this invention.

[0065] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0066] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0067] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0068] In this invention, unless otherwise stated, the term antigen-binding fragment is also known as "antibody fragment". Antibody fragment usually refers to an antigen-binding antibody fragment, which may include a part of a complete antibody, generally an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, biantibodies, linear antibodies, single-chain antibody molecules, etc.

[0069] The terms "complementarity-determining region" or "CDR" or "CDR sequence" refer to the amino acid sequence in an antibody responsible for antigen binding. For example, it typically includes amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable region, and amino acid residues near 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)).

[0070] Without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The sequence of the variants described in the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are shown in N-terminus to C-terminus arrangement.

[0071] In this paper, the terms “identity,” “homology,” or “similar phase” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0072] As previously described, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-TIGIT antibodies having modified glycosylation patterns. In some applications, modification to remove unwanted glycosylation sites may be useful, or to antibodies lacking a fucose moiety on the oligosaccharide chain to, for example, enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0073] As used herein, the term "functional fragment" specifically refers to antibody fragments such as Fv, scFv (sc stands for single-chain), Fab, F(ab')2, Fab', scFv-Fc fragments, or diabody fragments, or any fragment that should have its half-life increased by chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated," or "PEGylated") (referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG fragments) ("PEG" stands for polyethylene glycol), which have TIGIT binding activity. Preferably, the functional fragment will consist of or contain a portion of the heavy chain variable region or light chain variable region of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least 1 / 100th of the affinity for TIGIT, and more preferably at least 1 / 10th. This functional fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids from the antibody sequence from which it is derived.

[0074] As previously described, one aspect of the present invention provides an antibody or an antigen-binding fragment thereof. According to embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0075] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO: 1, 2, and 3 or amino acid sequences having at least 80% identity with SEQ ID NO: 1, 2, and 3; and

[0076] The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO: 4, 5, and 6, or amino acid sequences having at least 80% identity with 4, 5, and 6, respectively.

[0077] In a preferred embodiment of the present invention, to further improve the bioacceptability of the antibody, it can be humanized, i.e., the antibody is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to a recombinant antibody obtained by replacing the constant region amino acid sequence of a monoclonal antibody from one species (e.g., mouse) with the constant region of an antibody from another species (e.g., human) using recombinant DNA technology. The term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acid sequences of the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the non-CDR amino acid sequences of the constant and variable regions of an antibody from another species (e.g., human). In other words, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all non-CDR amino acid sequences of the constant and variable regions are humanized, it is called a humanized antibody. The humanization method can be performed using conventional antibody engineering techniques, and will not be elaborated further here.

[0078] In another aspect of the invention, a composition is provided. According to embodiments of the invention, the composition comprises: the aforementioned antibody or its antigen-binding fragment, a nucleic acid molecule, an expression vector, and recombinant cells.

[0079] The compositions provided by this invention contain antibodies or antigen-binding fragments thereof as described above, nucleic acid molecules, expression vectors, and recombinant cells. In some embodiments, the compositions comprise combinations that are separate in time and / or space, provided they can work together to achieve the objectives of this invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0080] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, or recombinant cells of the present invention can also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit TIGIT activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with upstream or downstream signal transduction of TIGIT.

[0081] Typically, the antibody or its antigen-binding fragment is administered in an effective amount, that is, an amount sufficient to achieve the desired therapeutic and / or preventative effect. For example, an amount that causes prevention or relief of symptoms associated with the disease being treated, such as a TIGIT-related disease. The effective amount of the composition administered to the subject will depend on the type and severity of the disease, as well as on individual characteristics such as general health status, age, sex, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, factors that a person skilled in the art will be able to determine the appropriate dosage based on.

[0082] In another aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises: the aforementioned antibody or its antigen-binding fragment, a nucleic acid molecule, an expression vector, recombinant cells, or a composition, and the medicament is used to treat malignant tumors.

[0083] The medicament provided by this invention comprises a therapeutic agent and an antibody or antigen-binding fragment thereof, as described above, conjugated to the therapeutic agent. The conjugation of the antibody or antigen-binding fragment thereof to the therapeutic agent can be in a conventional manner.

[0084] In another aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises the antibody or antigen-binding fragment thereof described in the first aspect of the invention.

[0085] In another aspect of the invention, the invention provides for the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit. According to an embodiment of the invention, the kit is used for the detection of TIGIT.

[0086] The kit for detecting TIGIT in a sample provided by this invention contains the antibody or its antigen-binding fragment as described above. The sample can be tissue from a patient suffering from a TIGIT-mediated disease (particularly patients with transplant rejection, autoimmune diseases, infectious diseases, or cancer, more preferably patients with at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer). The kit may also include reagents conventionally used for detecting TIGIT, such as coating solutions.

[0087] This invention also provides the use of the antibody or its antigen-binding fragment as described above in the preparation of reagents for detecting TIGIT in samples. As previously mentioned, the sample can be tissue from a patient suffering from a TIGIT-mediated disease, which will not be elaborated further here. The antibody or its antigen-binding fragment of this invention has good affinity for TIGIT and can effectively detect TIGIT in samples.

[0088] The present invention also provides the use of the antibody or its antigen-binding fragment in the preparation of a medicament for the prevention and / or treatment of TIGIT-mediated diseases. Preferably, the TIGIT-mediated diseases are transplant rejection, autoimmune diseases, infectious diseases, or cancer. More preferably, the cancer is a TIGIT-expressing cancer. Further preferably, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer. Further preferably, the infectious diseases include, but are not limited to, HIV infection and / or hepatitis B virus infection.

[0089] The present invention also relates to a method for preventing and / or treating TIGIT-mediated diseases (as described above), the method comprising: administering an effective amount of at least one of the antibodies of the present invention or antigen-binding fragments thereof, immunoconjugates, and compositions thereof to a patient. The administration may be by oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal administration.

[0090] In this invention, "patient" or "subject" generally refers to mammals, such as primates and / or rodents, especially humans or rats.

[0091] Those skilled in the art can clone DNA molecules encoding the antibodies or antigen-binding fragments described in this invention into vectors (especially expression vectors), transform them into host cells, and obtain the antibodies or antigen-binding fragments through induced expression. Therefore, this invention also provides nucleic acid molecules encoding the aforementioned antibodies or antigen-binding fragments and recombinant cells containing such nucleic acids. The nucleic acid is preferably an expression cassette obtained by genetic engineering methods.

[0092] Expression vectors can refer to cloning vectors or recombinant vectors. They can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). Commonly used plasmids include pSeTag2, PEE14, and pMH3.

[0093] The expression vector of the present invention may contain DNA sequences encoding the heavy chain variable region, light chain variable region, and / or constant region of the antibody. However, two expression vectors may also be constructed separately, one containing the heavy chain variable region and the constant region, and the other containing the light chain variable region and the constant region, and co-transfected into mammalian cells. In a preferred embodiment, the expression vector further contains a promoter and a DNA sequence encoding a secretion signal peptide, as well as at least one drug resistance gene for screening.

[0094] The recombinant cells described in this invention can be prokaryotic host cells, eukaryotic host cells, or bacteriophages. The prokaryotic host cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic host cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, and more preferably BHK cells, CHO cells, NSO cells, or COS cells.

[0095] The nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention. Based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can readily obtain the corresponding nucleotide sequences.

[0096] In this paper, antibody affinity maturation, as described in this invention, refers to a normal immune functional state in the body. In humoral immunity, the average affinity of antibodies produced in a secondary immune response is higher than that of the primary immune response; this phenomenon is called antibody affinity maturation. This functional state of the body is the result of long-term evolution and continuous adaptation to the external environment, and it is of great significance for the body's defense and maintenance of its own immune surveillance. This invention utilizes in vitro antibody affinity maturation technology; specifically, it involves using molecular biology techniques to mutate the CDR amino acids of antibodies, and then screening from a library of mutated antibodies to obtain antibodies with significantly enhanced affinity.

[0097] In this paper, H401LV2 represents the TIGIT antibody prepared in this invention.

[0098] In this paper, Ka and kon have the same meaning, representing the binding constant. Kd and koff have the same meaning, representing the dissociation constant.

[0099] In this article, Jurkat cells refer to an immortalized human T lymphocyte line of suspension cells used to study acute T-cell leukemia, T-cell signaling, and the expression of various chemokine receptors, particularly HIV, in susceptible viral entry.

[0100] Embodiments of the present invention will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0101] It should be noted that the terms "plasmid" and "vector" used in the following implementation have the same meaning and can be used interchangeably.

[0102] The maternal antibody Hu4A501 used in the following examples refers to a humanized TIGIT antibody developed by our research group, the sequence of which can be found in CN109384846B.

[0103] The Tiragolumab analog used is an analog of the anti-human TIGIT antibody Tiragolumab developed by Roche, which has the same amino acid sequence as Tiragolumab and was purchased from BioNTech.

[0104] In the following examples, hIgG1 refers to the constant region of wild-type human IgG1, and antibodies of this type typically have ADCC (antibody-mediated cytotoxicity) function; hIgG1LALA refers to the constant region of human IgG1 with L234A and L235A mutations, which eliminate ADCC function; hIgG4S228P refers to the constant region of human IgG4 with S228P mutations. Because wild-type IgG4 exhibits arm exchange, S228P mutations are typically performed in antibody engineering to eliminate this effect.

[0105] Example 1: Antibody Affinity Maturation

[0106] To improve the druggability of the antibody and obtain a high-affinity TIGIT antibody, the inventors performed affinity maturation on the developed humanized TIGIT antibody Hu4A501 (CN109384846B) (using FASEBA technology, completed by Genscript Biotech). The antigen used for affinity maturation was the recombinant TIGIT extracellular Fc fusion protein (TIGIT-Fc) (amino acid sequence shown in SEQ ID NO:9). The resulting matured TIGIT antibody H401LV2 heavy chain variable region and light chain variable region are shown in SEQ ID NO:7 and 8.

[0107] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGR TTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTV SS(SEQ ID NO:7),

[0108] DIVMTQSPSSMTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTG VPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELK(SEQ ID NO:8),

[0109] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLELEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:9).

[0110] Example 2: Antibody Affinity Detection

[0111] The Biacore method is a recognized objective method for detecting the affinity and binding kinetics between proteins. The affinity and binding kinetics of the TIGIT antibody of this invention were characterized by analysis using the Biacore T200.

[0112] The TIGIT extracellular Fc fusion protein (TIGIT-Fc) was covalently linked to a CM5 (GE) chip using a standard amino-coupled method. A series of concentration gradients of TIGIT antibody diluted in PBS buffer were then injected into the chip for each cycle, followed by regeneration with 10 mM NaOH solution. Antigen-antibody binding kinetics were tracked for 3 minutes and dissociation kinetics for 10 minutes. Data were analyzed using GE's BIAevaluation software with a 1:1 (Langmuir) binding model. The Ka (kon), Kd (koff), and KD values ​​determined by this method are shown in Table 1 below.

[0113] Table 1: Affinity data of maternal antibody (Hu4A501) and maturated antibody H401LV2

[0114] Ka(1 / Ms) Kd(1 / s) KD(M) Hu4A501 9.89E+04 5.01E-04 5.06E-09 h401LV2 1.97E+05 1.00E-06 5.08E-12

[0115] Note: Ka represents the binding constant (the larger the value, the stronger the affinity); Kd represents the dissociation constant (the smaller the value, the stronger the affinity), reflecting the affinity of the compound for the target; KD represents Kd / Ka, which is the affinity constant.

[0116] Example 3: TIGIT antibody ELISA binding assay

[0117] According to an embodiment of the present invention, the binding characteristics of TIGIT antibody are detected using an ELISA assay. The His-tagged fusion protein of the extracellular region of TIGIT is coated into a 96-well plate. After adding the antibody, the binding characteristics between the antibody and the TIGIT protein are determined based on the signal strength. The specific experimental steps are as follows:

[0118] 1. Antibody preparation

[0119] Antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with the pcDNA3.4 vector (synthesized by Nanjing GenScript, containing Hu4A501 light chain i.e., Hu4A501-hK (amino acid sequence as shown in SEQ ID NO: 17), Hu4A501 heavy chain H401LV2-hIgG1 (amino acid sequence as shown in SEQ ID NO: 18), H401LV2 light chain i.e., H401LV2-hK (amino acid sequence as shown in SEQ ID NO: 19), H401LV2 heavy chain H401LV2-hIgG1 (amino acid sequence as shown in SEQ ID NO: 20), H401LV2-hIgG1LALA (amino acid sequence as shown in SEQ ID NO: 21), and H401LV2-hIgG4S228P (amino acid sequence as shown in SEQ ID NO: 22).

[0120] DIVMTQSPSSMSTSVGDRVTITCRASQDVFTAVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:17)

[0121] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKPEKSCDKTH TCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPG(SEQ ID NO:18)

[0122] DIVMTQSPSSMSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 19)

[0123] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20)

[0124] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:21)

[0125] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQG TLLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:22)

[0126] One day before transfection, the cell density of ExpiCHO-S cells was adjusted to (3-4) × 10⁻⁶. 6 / mL, cultured overnight at 37℃, 8% CO2, and 95 rpm with shaking. On the day of transfection, cells grew to 7 × 10⁹ / mL. 6 ~1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / mL using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6Transfect the cells with the above-mentioned pcDNA3.4 plasmid carrying heavy and light chains (light-heavy chain plasmid ratio 1:1) and ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129) into ExpiCHO-S cells at 37℃, 8% CO2, and 95 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32℃, 5% CO2, and 95 rpm with shaking. On day 5 post-transfection, add another 8 mL of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability changes daily. Harvest cells by centrifugation when cell viability drops below 80% or after 10-14 days of culture. The supernatant from the expression was filtered through a 0.45 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselectprism A protein A affinity chromatography column (purchased from Suzhou Nanomicro). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 95%. The results showed that the above-mentioned antibody was finally obtained.

[0127] 2. ELISA assay to detect the binding characteristics of TIGIT antibody

[0128] 1. Dilute the human and cynomolgus monkey TIGIT-His tag fusion protein (purchased from Acro) with PBS buffer to 2 μg / ml, add 100 μl / well to a 96-well plate, and incubate overnight at 4°C.

[0129] 2. Remove the PBS buffer from the 96-well plate in step 1, wash the plate 6 times with PBST buffer (pH 7.2; PBST contains 0.1% Tween 20 in total volume of buffer), add 200 μl / well of PBS buffer (containing 10% volume of BSA (bovine serum albumin)), and incubate at 37°C for 2 hours to block.

[0130] 3. Remove the PBS buffer from step 2, wash the plate 6 times with PBST buffer, add 100 μl / well of the TIGIT antibody to be tested diluted to 10000, 2000, 400, 80, 16, 3.2, 0.64, and 0.128 ng / ml with PBST buffer (containing 0.05% volume of BSA), and incubate at 37°C for 1 h.

[0131] 4. Remove the buffer from step 3, wash the plate 6 times with PBST, and then dilute the HRP (horseradish peroxidase) labeled anti-human IgG antibody (secondary antibody, purchased from Jacksonlab) with PBST buffer (containing 0.05% volume BSA) at 100 μl / well and incubate at 37°C for 1 h.

[0132] 5. Wash the 96-well plate from step 4 six times with PBST buffer, add 80 μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then add 80 μl / well 4M sulfuric acid to terminate the reaction.

[0133] 6. Use an ELISA reader to read the absorbance of the 96-well plate at 450 nm in step 5.

[0134] The results are as follows Figure 1 As shown, the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) prepared in this invention can bind to the TIGIT protein, and the binding is stronger than that of the parent antibody Hu4A501 and the Tiragolumab analog (purchased from Baiying Biotechnology).

[0135] Example 4: TIGIT antibody ELISA blocking assay

[0136] According to an embodiment of the present invention, the binding characteristics of TIGIT antibody are detected using an ELISA assay. The His-tagged fusion protein of the extracellular region of TIGIT is coated into a 96-well plate. After adding antibody and CD155-Biotin, the antibody's ability to block TIGIT / CD155 binding is determined based on the signal strength. The specific steps are as follows:

[0137] 1. Dilute the human and cynomolgus monkey TIGIT-His tag fusion protein (purchased from Acro) with PBS buffer to 2 μg / ml, add 100 μl / well to a 96-well plate, and incubate overnight at 4°C.

[0138] 2. Remove the PBS buffer from the 96-well plate in step 1, wash the plate 6 times with PBST buffer (pH 7.2; PBST buffer contains 0.1% Tween 20), and then add 200 μl / well of PBS buffer (containing 10% BSA).

[0139] (Bovine serum albumin) 10% BSA, incubated at 37°C for 2 hours for blocking.

[0140] 3. Remove the PBS buffer blocking solution from step 2, wash the plate 6 times with PBST buffer, add 100 μl / well of TIGIT antibody diluted to the appropriate concentration with PBST buffer (containing 0.05% volume BSA) / 0.05% BSA, and incubate at 37°C for 1 h.

[0141] 4. Remove the buffer solution from step 3 of the reaction system, wash the plate 6 times with PBST, and then dilute the HRP (horseradish peroxidase) labeled anti-human IgG antibody secondary antibody (secondary antibody, purchased from Jacksonlab) with PBST buffer (containing 0.05% volume BSA) / PBST / 0.05% BSA at 100 μl / well, and incubate at 37°C for 1 h.

[0142] 5. After washing the 96-well plate from step 4 six times with PBST buffer, add 80 μl / well TMB (tetramethylbenzidine), incubate at room temperature for 3 min, and then add 80 μl / well 4M sulfuric acid to terminate the reaction.

[0143] 6. Use an ELISA reader to read the absorbance of the 96-well plate from step 5 at 450 nm.

[0144] The results are as follows Figure 2 As shown, the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) prepared in this invention can block TIGIT / CD155 binding, and its blocking ability is no weaker than that of Tiragolumab analogs (purchased from Bio-Tech).

[0145] Example 5: Experiment on TIGIT antibody binding to 293T-TIGIT

[0146] According to an embodiment of the present invention, the binding characteristics of TIGIT antibody were detected by flow cytometry. TIGIT protein (denoted as 293T-TIGIT) was overexpressed in HEK293T cells (ATCC number CRL-3216). After adding the antibody (please specify the antibody added here), the binding characteristics of the chimeric antibody and TIGIT were determined based on the signal strength. The specific steps are as follows:

[0147] 1. HEK293T cells were accumulating at a rate of 5 × 10⁻⁶. 5 Cells / wells were added to six-well plates and cultured overnight in DMEM medium without antibiotics.

[0148] 2. Before transfection, discard the culture medium and add 1 ml of fresh DMEM medium without antibiotics. Add the pLVX-EF1a-TIGIT-IRES-puro vector (the pLVX-EF1a-IRES-puro vector has a TIGIT protein sequence (SEQ ID NO:11) inserted between the EcoRI and BamHI restriction sites) to 200 μl of serum-free DMEM medium, along with pMD2G and psPAX2 vectors (3 μg of the three vectors in total), in a 2:1:1 ratio.

[0149] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFL EVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSSAPSPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG(SEQ ID NO: 11).

[0150] 3. Add 12 μg of polyetherimide (PEI, Polysciences Ltd.) to the culture medium in step 2. Mix well and let stand for 16 min. Then add all the liquid to a six-well plate containing HEK293T cells and incubate for 6 h.

[0151] 4. Discard the culture medium in the six-well plate and add fresh complete DMEM medium for further culture. 48 h after transfection, collect the cell culture supernatant and filter it through a 0.45 μm filter (purchased from Millipore) to obtain the viral supernatant.

[0152] 5. Add all the viral supernatant from step 4 to a solution containing 1×10 4 Polybrene (purchased from Sigma) was added to a 6-well plate of HEK293T cells at a final concentration of 4 μg / ml and cultured for 12 h.

[0153] 6. Discard the supernatant from step 5 and add fresh complete DMEM medium. The resulting cells are 293T-TIGIT cells.

[0154] 7. Dilute 293T-TIGIT cells to 2×10⁻⁶ with PBS. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of mouse serum, and block at 4℃ for 30min.

[0155] 8. Add TIGIT antibody at different concentration gradients (maximum 8 μg / ml, 5-fold gradient dilution, 8 gradients) to the EP tube in step 7 and incubate at 4°C for 30 min.

[0156] 9. Add 1 ml of PBS to the EP tube from step 8, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100 μl / tube of PBS, add 1 μl / tube of Alexa-647-labeled mouse anti-human Fc antibody (secondary antibody, purchased from Jacksonlab), and incubate at 4°C in the dark for 30 min.

[0157] 10. Wash the cells from step 9 twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS buffer and analyze using flow cytometry.

[0158] The results are as follows Figure 3 As shown, the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) of the present invention can bind to 293T-TIGIT.

[0159] Example 6: Detection of TIGIT antibody blocking ability

[0160] TIGIT antibodies block the binding of TIGIT to its ligand CD155 by binding to the extracellular domain of TIGIT. In this embodiment of the invention, flow cytometry was used to detect the blocking effect of TIGIT antibodies on CD155 binding to 293T-TIGIT. The specific steps are as follows:

[0161] 1. Dilute 293T-TIGIT cells (same as in Example 5) to 2×10⁻⁶ with PBS. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of mouse serum, and block at 4℃ for 30min.

[0162] 2. Add up to 200 μg / ml of TIGIT antibody and CD155-mIgG2a-Fc (purchased from Acro) in a 5-fold serial dilution (a total of 10 dilutions) to the EP tube from step 1, and incubate at 4°C for 30 min.

[0163] 3. Add 0.1 μg of APC-labeled goat anti-mouse IgG2a secondary antibody (purchased from Biolegend) to the EP tubes from step 2, and incubate at 4°C for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl of PBS per tube and analyze using flow cytometry.

[0164] The results are as follows Figure 4 As shown, the TIGIT antibodies H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) of the present invention, which have different constant regions, can all block the binding of CD155-mIgG2a to 293T-TIGIT, and their blocking ability is no weaker than that of Tiragolumab analogs (purchased from Bio-Tech).

[0165] Example 7: TIGIT antibody promotes Jurkat T cell activation assay

[0166] Jurkat cells were co-incubated with U-937 tumor cells, and CD3 and TIGIT antibodies were added. The IL-2 content in the supernatant was measured. The activation effect of Jurkat cells was determined based on the strength of the IL-2 signal after antibody addition. The specific operation steps are as follows:

[0167] 1. Dilute the OKT3 antibody (purchased from Biolegend) to 0.2 μg / ml with PBS, add it to a 96-well plate, react overnight, and discard the supernatant.

[0168] 2. Dilute Jurkat cells to 1×10⁻⁶ using complete 1640 medium. 6 / ml, added to 96-well plates at a volume of 100μl / tube; U-937 cells were diluted to 1.25×10 with complete 1640 medium. 6 / ml, add 80μl / tube to a 96-well plate; dilute TIGIT antibody to 100μg / ml with complete 1640 medium and add 20μl / well to a 96-well plate. Incubate at 37℃ in a 5% CO2 incubator for 24h, and detect IL-2 in the supernatant using a CBA kit (purchased from BD).

[0169] The results are as follows Figure 5 As shown, the affinity-matured TIGIT antibody H401LV2-hlgG1LALA of the present invention has a better ability to promote Jurkat T cell activation than the parent antibody Hu4A501-hlgG1LALA.

[0170] Example 8: TIGIT antibody binding to CHO-K1-TIGIT experiment

[0171] This invention utilizes flow cytometry to detect the binding properties of TIGIT antibodies. TIGIT protein (labeled CHO-K1-TIGIT) was overexpressed in CHO-K1 cells (ATCC number CCL-61). The binding properties of the chimeric antibody and TIGIT were determined based on the strength of the signal after antibody addition.

[0172] 1. Antibody preparation: The pcDNA3.4 vector (synthesized by Nanjing GenScript, 4.1D3 light chain i.e. 4.1D3-mK (amino acid sequence as shown in SEQ ID NO: 10), 4.1D3 heavy chain i.e. 4.1D3-mIgG2a (amino acid sequence as shown in SEQ ID NO: 11), 22G2 light chain i.e. 22G2-mK (amino acid sequence as shown in SEQ ID NO: 12), 22G2 heavy chain i.e. 22G2-mIgG2a (amino acid sequence as shown in SEQ ID NO: 13), H401LV2 light chain i.e. H401LV2-mK (amino acid sequence as shown in SEQ ID NO: 14), and H401LV2 heavy chain i.e. H401LV2-mIgG2a (amino acid sequence as shown in SEQ ID NO: 15) of the above-mentioned antibody were transiently transfected into ExpiCHO-S cells (Gibco, catalog number A29127) to prepare antibodies.

[0173] DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEI KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO: 10),

[0174] EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:11),

[0175] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLFTFGPGTKVDIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQID NO:12),

[0176] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGIYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYCARDYYVSGNYYNVDYYFFGVDVWGQGTTVVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSTWPSQSITCNVAHPASSTKVDKKIEPRGP TIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKFSRTPGK(SEQ ID NO:13)。

[0177] DIVMTQSPSSMSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO: 14)

[0178] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGT LLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:15)

[0179] One day before transfection, the cell density of ExpiCHO-S cells was adjusted to (3-4) × 10⁻⁶. 6 / mL, cultured overnight at 37℃, 8% CO2, and 95 rpm with shaking. On the day of transfection, cells grew to 7 × 10⁹ / mL. 6 ~1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / mL using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6Transfect the cells with the above-mentioned pcDNA3.4 plasmid carrying heavy and light chains (light-heavy chain plasmid ratio 1:1) and ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129) into ExpiCHO-S cells at 37℃, 8% CO2, and 95 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32℃, 5% CO2, and 95 rpm with shaking. On day 5 post-transfection, add another 8 mL of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability changes daily. Harvest cells by centrifugation when cell viability drops below 80% or after 10-14 days of culture. The supernatant from the expression was filtered through a 0.45 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselectprism A protein A affinity chromatography column (purchased from Suzhou Nanomicro). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 95%. The results showed that the above-mentioned antibody was finally obtained.

[0180] 2. Flow cytometry detection of the binding characteristics of chimeric antibodies to TIGIT

[0181] 1. HEK293T cells were accumulating at a rate of 5 × 10⁻⁶. 5 Cells / wells were added to six-well plates and cultured overnight in DMEM medium without antibiotics.

[0182] 2. Before transfection, discard the culture medium and add 1 ml of fresh DMEM medium without antibiotics. Add the pLVX-EF1a-TIGIT-IRES-puro vector (the pLVX-EF1a-IRES-puro vector has TIGIT protein inserted between the EcoRI and BamHI restriction sites) pMD2G and psPAX2 vector (3 μg of the three vectors in total) to 200 μl of serum-free DMEM medium in a ratio of 2:1:1.

[0183] 3. Add 12 μg of polyetherimide (PEI, Polysciences Ltd.) to the culture medium in step 2, mix well and let stand for 16 min, then add all the liquid to a six-well plate containing CHO-K1 cells.

[0184] 4. Incubate the six-well plate from step 3 for 6 hours, discard the culture medium, and add fresh complete DMEM culture medium for further incubation.

[0185] 5. 48 hours after transfection, collect the cell culture supernatant and filter it using a 0.45μm filter (purchased from Millipore). The filtrate obtained is the virus supernatant.

[0186] 6. Add all of the virus supernatant to a solution containing 1×10 4 Polybrene (purchased from Sigma) was added to 6-well plates containing 293T cells at a final concentration of 4 μg / ml and cultured for 12 h.

[0187] 7. Discard the supernatant and add fresh complete DMEM medium for culturing. The resulting cells are CHO-K1-TIGIT cells.

[0188] 8. Dilute CHO-K1-TIGIT cells to 2 × 10⁻⁶ with PBS. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of mouse serum, and block in a 4℃ refrigerator for 30min.

[0189] 9. Add TIGIT antibody at different concentration gradients to the EP tubes from step 8 and incubate at 4°C for 30 min. Add 1 ml of PBS to the EP tubes, centrifuge at 3500 rpm for 5 min at 4°C, and discard the supernatant.

[0190] 10. Resuspend the cells in PBS again, centrifuge and discard the supernatant. Resuspend the cells in 100 μl / tube of PBS, add 1 μl / tube of Alexa-647-labeled mouse anti-human Fc antibody (secondary antibody, purchased from Jacksonlab), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge and discard the supernatant.

[0191] 11. Resuspend the cells in 200 μl / tube of PBS and then perform flow cytometry.

[0192] The results are as follows Figure 6 As shown, the TIGIT antibody H401LV2-mlgG2a of the present invention can bind CHO-K1-TIGIT, and its binding ability is superior to that of the 4.1D3 and 22G2 antibodies.

[0193] Example 9: Detection of TIGIT antibody blocking ability

[0194] TIGIT antibodies block the binding of TIGIT to its ligand CD155 by binding to the extracellular region of TIGIT. This invention utilizes flow cytometry to detect the blocking effect of TIGIT antibodies on CD155 binding to CHO-K1-TIGIT. The specific steps are as follows:

[0195] 1. Dilute CHO-K1-TIGIT cells (same as in Example 8) with PBS to a concentration of 2 × 10⁻⁶. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of mouse serum, and block at 4℃ for 30min.

[0196] 2. Add TIGIT antibody (30, 10, 3, 1, 0.1, 0.01, 0.001 μg / ml) and CD155-mIgG2a-Fc (purchased from Acro), and incubate at 4°C for 30 min. Add 0.1 μg / tube of APC-labeled goat anti-mouse IgG2a (secondary antibody, purchased from Biolegend), and incubate at 4°C for 30 min.

[0197] 3. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS, and finally perform flow cytometry analysis.

[0198] The results are as follows Figure 7 As shown, the antibody H401LV2-mlgG2a of the present invention can block the binding of CD155 to CHO-K1-TIGIT, and its blocking ability is stronger than that of the 4.1D3 antibody.

[0199] Example 10: Effects of H401LV2 antibody on anticancer activity in mice

[0200] In vivo efficacy experiments were conducted to detect the effect of the affinity-matured TIGIT antibody H401LV2 on the anticancer function of hTIGIT transgenic mice. The specific steps are as follows:

[0201] 1. Antibody preparation

[0202] Antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with the pcDNA3.4 vector (synthesized by Nanjing GenScript, H401LV2 light chain i.e. H401LV2-mK (amino acid sequence as shown in SEQ ID NO: 14), H401LV2 heavy chain H401LV2-mIgG2a (amino acid sequence as shown in SEQ ID NO: 15), and H401LV2-mIgG2aD265A (amino acid sequence as shown in SEQ ID NO: 16) containing the heavy chain and light chain nucleotide sequences of the above antibodies.

[0203] QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGT LLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:16)

[0204] One day before transfection, the cell density of ExpiCHO-S cells was adjusted to (3-4) × 10⁻⁶. 6 / mL, cultured overnight at 37℃, 8% CO2, and 95 rpm with shaking. On the day of transfection, cells grew to 7 × 10⁹ / mL. 6 ~1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / mL using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6Transfect the cells with the above-mentioned pcDNA3.4 plasmid carrying heavy and light chains (light-heavy chain plasmid ratio 1:1) and ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129) into ExpiCHO-S cells at 37℃, 8% CO2, and 95 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32℃, 5% CO2, and 95 rpm with shaking. On day 5 post-transfection, add another 8 mL of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability changes daily. Harvest cells by centrifugation when cell viability drops below 80% or after 10-14 days of culture. The supernatant from the expression was filtered through a 0.45 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselectprism A protein A affinity chromatography column (purchased from Suzhou Nanomicro). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 95%. The results showed that the above-mentioned antibody was finally obtained.

[0205] 2. Detection of anti-cancer function of H401LV2 in hTIGIT transgenic mice. 1. Day 0, hTIGIT tumor-bearing mice (purchased from Southern Model Animals) were injected with 2×10⁻⁶ cells per mouse. 5 MC38 cells ( Figure 8 Or inject 1×10⁻⁶ mice. 6 LLC cells ( Figure 9 The mice were then randomly divided into groups.

[0206] 2. On days 4, 7, 10, and 13, mice were intraperitoneally injected with the aforementioned affinity maturation antibody, 250 μg per mouse;

[0207] 3. Measure the tumor volume every 3 days after the above-mentioned antibody is injected.

[0208] The results are as follows Figure 8 As shown, H401LV2-mIgG2a, which has ADCC effector function, has better therapeutic effect in the MC38 colorectal cancer model; Figure 9 As shown, H401LV2-mIgG2aD265A, which does not have ADCC effect function, has better efficacy in LLC lung cancer model.

[0209] The experimental results above demonstrate that the high-affinity antibody obtained in this invention can bind to TIGIT. By blocking the interaction between TIGIT and CD155, it activates the anti-cancer mechanism of immune cells.

[0210] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0211] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0212] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, include: The heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, the heavy chain variable region CDR2 shown in SEQ ID NO: 2, the heavy chain variable region CDR3 shown in SEQ ID NO: 3, the light chain variable region CDR1 shown in SEQ ID NO: 4, the light chain variable region CDR2 shown in SEQ ID NO: 5, and the light chain variable region CDR3 shown in SEQ ID NO:

6. The antibody or its antigen-binding fragment specifically recognizes TIGIT.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a humanized antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain framework region sequence and a light chain framework region sequence, wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a primate antibody, or a mutant thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a primate-derived antibody and a murine antibody or a mutant thereof.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG1 antibody, IgG2a antibody or their mutants, or human IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody or their mutants.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is at least one of single-chain antibody, multi-polymer antibody, CDR transplantation antibody, Fab antibody, and Fv antibody.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 7.

9. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 8.

10. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that, include: The expression vector according to claim 9 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the antibody or its antigen-binding fragment.

11. The method according to claim 10, characterized in that, The cells in question are eukaryotic cells.

12. A non-plant recombinant cell, characterized in that, The recombinant cells express the antibody or antigen-binding fragment of any one of claims 1-7, and carry the nucleic acid molecule of claim 8 or the expression vector of claim 9.

13. A composition, characterized in that, include: The antibody or its antigen-binding fragment according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 12.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 12, or the composition according to claim 13 in the preparation of a medicament for the treatment of lung cancer and colorectal cancer.

15. A drug, characterized in that, include: The drug is used to treat lung cancer and colorectal cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 12, or the composition according to claim 13.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7 in the preparation of a kit for detecting TIGIT.

17. A reagent kit, characterized in that, The kit contains the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7.

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