High-affinity TIGIT antibodies and their applications

Abstract

The present application provides an anti-TIGIT antibody or antigen-binding fragment thereof and applications thereof, which comprises heavy chain variable region CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, and light chain variable region CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 4, 5, and 6.

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies. Specifically, the present invention relates to high-affinity TIGIT antibodies and applications thereof. More specifically, the present invention relates to antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, methods for preparing antibodies or antigen-binding fragments thereof, recombinant cells, compositions and uses thereof, drugs and uses thereof, and methods for treating malignant tumors. [Background technology]

[0002] Malignant tumors are diseases that seriously threaten the life and health of humankind. Currently available treatments for malignant tumors include surgical resection, radiation therapy, chemotherapy, small molecule targeted therapy, immune checkpoint therapy, and cell and gene therapy, but these only have limited effectiveness in a small proportion of malignant tumor patients. Malignant tumors remain a serious problem affecting the life and health of humankind.

[0003] In recent years, immunotherapy has made remarkable progress in the field of tumor treatment, with particular attention being paid to immune checkpoint blockade therapies, such as anti-CTLA-4 and anti-PD-1 or PD-L1 antibodies. Blocking the binding of inhibitory receptors on the T cell surface to their ligands blocks the transmission of inhibitory signals, corrects immunosuppression mediated by the immunosuppressive microenvironment, and restores the antitumor capabilities of T cells in the tumor microenvironment. This has resulted in high response rates in the treatment of various metastatic, advanced malignancies (including metastatic melanoma, non-small cell lung cancer, and renal carcinoma). However, even with the currently broadest approved indication, the overall response rate of PD-1 / L1 therapy is only 30%, and it cannot be ignored that many more patients are missing out on its benefits.

[0004] However, not all malignant tumor patients respond well to PD-1, PD-L1, or CTLA-4 blockade therapy; only 10%-30% of patients achieve long-term, durable responses to PD-1 or PD-L1 antibody therapy, with the majority failing to respond. Meanwhile, due to the relatively heterogeneous expression of immune checkpoint ligands in tumors and tumor-infiltrating lymphocytes, a single type of immune checkpoint therapy cannot be applied to all patients, and many patients fail to benefit from it. Meanwhile, some patients who receive immune checkpoint therapy experience tumor recurrence and develop resistance to the immune checkpoint therapy, resulting in a lack of therapeutic efficacy even with continued administration, i.e., secondary resistance. For these two reasons, the development of immune checkpoint antibodies targeting more targets is necessary.

[0005] T cell immunoglobulin protein and ITIM (Immunoreceptor tyrosine-based inhibitory motif) domain protein (TIGIT) are important immune checkpoint proteins that are primarily expressed on the surface of natural killer (NK) cells, activated CD8+ T and CD4+ T cells, regulatory T cells (Tregs), and follicular helper T cells (Tfh). The ligands recognized by TIGIT, CD155 and CD112, are primarily expressed on monocytes, macrophages, dendritic cells (DCs), T cells, B cells, and many nonhematopoietic cells, including various histological types of tumor cells. The affinity of the TIGIT-binding ligand, PVR (polivirus receptor, CD155), is significantly higher than that of its competing receptors, CD226 and CD96. Binding of TIGIT to its ligand 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 (e.g., 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 TIGIT is highly expressed on the surface of tumor-infiltrating T cells in lung cancer patients, and its expression is significantly correlated with PD-1 expression. TIGIT expression on the surface of tumor-infiltrating NK cells in colon cancer patients is significantly higher than that on peripheral blood NK cells. In multiple mouse tumor models, antibody blockade of TIGIT can suppress tumor growth and metastasis, while blockade of TIGIT and PD-1 can effectively treat tumors. Therefore, there is a particular need for the development of therapeutic TIGIT antibodies. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to solve at least to some extent one of the technical problems in the related art. [Means for solving the problem]

[0008] To inhibit tumor growth and metastasis and the resulting problem of secondary resistance, the present invention provides a high-affinity TIGIT antibody and its application in the treatment of malignant tumors.

[0009] The antibody according to the present invention blocks the binding of TIGIT to its ligand CD155, thereby relieving TIGIT-mediated immunosuppression and promoting the anti-cancer function of immune cells.

[0010] Therefore, to achieve the above object, in a first aspect, the present invention provides an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 1, 2, and 3; and The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs: 4, 5, and 6, or amino acid sequences having at least 80% identity to 4, 5, and 6, respectively.

[0011] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof binds to TIGIT and blocks the binding of TIGIT to its ligand CD155, thereby relieving TIGIT-mediated immunosuppression and activating the immune cell anti-cancer mechanism, thereby further suppressing tumor growth and proliferation. Furthermore, the high specificity of the antibody or antigen-binding fragment thereof avoids the occurrence of secondary resistance problems.

[0012] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof may further include at least one of the following additional technical features:

[0013] According to an embodiment of the invention, the antibody or antigen-binding fragment thereof comprises: It further includes a heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, a heavy chain variable region CDR2 shown in SEQ ID NO:2, a heavy chain variable region CDR3 shown in SEQ ID NO:3, a light chain variable region CDR1 shown in SEQ ID NO:4, a light chain variable region CDR2 shown in SEQ ID NO:5, and a light chain variable region CDR3 shown in SEQ ID NO:6. GSSITSDYA (SEQ ID NO: 1), ITYSGRT (SEQ ID NO: 2), ARWGLLRRYFDY (SEQ ID NO: 3), QDVFNQ (SEQ ID NO: 4), SASFRYT (SEQ ID NO: 5), QQHYSTPLT (SEQ ID NO: 6).

[0014] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes TIGIT.

[0015] According to an embodiment of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:8; QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSS(SEQ ID NO:7), DIVMTQSPSSMSSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELK(SEQ ID NO:8).

[0016] According to an embodiment of the present invention, the antibody is a humanized antibody.

[0017] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region sequence and a light chain framework region sequence, and 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 mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a variant thereof.

[0018] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises 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 human-derived antibody, a primate-derived antibody, and a mouse-derived antibody or a variant thereof.

[0019] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a mouse IgG1 antibody, IgG2a antibody, or a mutant thereof, or a human IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, or a mutant thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is at least one of a single-chain antibody, a multimeric antibody, a CDR-grafted antibody, a Fab antibody, and an Fv antibody.

[0020] In a second aspect, the present invention provides a nucleic acid molecule, according to an embodiment of the present invention, encoding an antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0021] Antibodies or antigen-binding fragments thereof encoded by nucleic acid molecules according to embodiments of the present invention have stronger specificity, longer half-life, and higher efficacy, and can effectively treat or prevent malignant tumors at lower doses, with fewer toxic side effects and greater safety.

[0022] In a third aspect, the present invention provides an expression vector, which, according to an embodiment of the present invention, carries a nucleic acid molecule according to the second aspect of the present invention.

[0023] The expression vectors mentioned in the examples of the present invention can efficiently express the above-mentioned antibodies or antigen-binding fragments thereof in appropriate recipient cells, and the antibodies or antigen-binding fragments thereof designed in the present invention have stronger specificity and higher safety.

[0024] In a fourth aspect, the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the method comprises: introducing into a cell an expression vector according to the third aspect of the present invention; Culturing the cells under conditions suitable for protein expression and secretion, and obtaining the antibody or antigen-binding fragment thereof.

[0025] The inventors have discovered that the antibodies or antigen-binding fragments thereof prepared according to the embodiments of the present invention can be cultured more efficiently to produce highly pure antibodies or antigen-binding fragments thereof, and the operation steps are simpler and less costly.

[0026] According to an embodiment of the present invention, the cell is a eukaryotic cell.

[0027] In a fifth aspect, the present invention provides a recombinant cell, according to an embodiment of the present invention, which expresses an antibody or antigen-binding fragment thereof according to the first aspect of the invention and which carries a nucleic acid molecule according to the second aspect of the invention or an expression vector according to the third aspect of the invention.

[0028] According to some specific embodiments of the present invention, under appropriate conditions, the recombinant cells can efficiently express large amounts of antibodies or antigen-binding fragments thereof, and the antibodies or antigen-binding fragments thereof have stronger specificity, longer half-lives, and higher efficacy, and can deliver antibody drugs to target cells at lower doses, effectively treating or preventing malignant tumors, with fewer toxic side effects and greater safety.

[0029] In a sixth aspect, the present invention provides a composition, according to an embodiment of the present invention, comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, an expression vector according to the third aspect of the invention or a recombinant cell according to the fifth aspect of the invention.

[0030] According to an embodiment of the present invention, the composition of the present invention, when introduced into a living body, can achieve the same therapeutic effect as an antibody or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, and a recombinant cell.

[0031] In a seventh aspect, the present invention provides the use of the composition in the preparation of a medicament, which, according to an embodiment of the present invention, is used to treat or prevent malignant tumors.

[0032] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof can be used to prepare a drug, which has the same effect as the antibody or antigen-binding fragment thereof and is used to treat or prevent diseases such as malignant tumors.

[0033] According to an embodiment of the present invention, the malignant tumor includes 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.

[0034] In an eighth aspect of the present invention, the present invention proposes a medicament, according to an embodiment of the present invention, comprising an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, an expression vector according to the third aspect of the present invention, a recombinant cell according to the fifth aspect of the present invention or a composition according to the sixth aspect of the present invention, for use in treating malignant tumors.

[0035] The inventors have discovered that a drug containing an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a recombinant cell or a composition has excellent therapeutic effects in the treatment and prognosis of malignant tumors.

[0036] According to an embodiment of the present invention, the malignant tumor includes 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.

[0037] In a ninth aspect of the present invention, the present invention proposes the use of the antibody or antigen-binding fragment thereof in the preparation of a kit, which, according to an embodiment of the present invention, is used to detect TIGIT.

[0038] According to an embodiment of the present invention, the kit detects TIGIT more efficiently and accurately, saving time and research costs for clinical treatment.

[0039] In a tenth aspect, the present invention proposes a kit, which according to an embodiment of the present invention comprises an antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0040] The inventors have discovered that kits prepared based on antibodies or antigen-binding fragments thereof can be applied in scientific research to save time and costs in analyzing malignant tumors.

[0041] In an eleventh aspect, the present invention provides a method for treating or preventing malignant tumors. According to an embodiment of the present invention, the method comprises administering to a subject an antibody or antigen-binding fragment thereof according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect, a composition according to the sixth aspect, or a drug according to the eighth aspect. Administration of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell, composition, or drug according to any embodiment of the present invention can be used to treat or prevent malignant tumors.

[0042] According to an embodiment of the present application, the malignant tumor includes 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.

[0043] In a twelfth aspect of the present invention, the present invention proposes the use of an antibody or antigen-binding fragment thereof according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect, a composition according to the sixth aspect, or a drug according to the eighth aspect in the treatment or prevention of malignant tumors. According to embodiments of the present invention, the antibody or antigen-binding fragment, recombinant protein nucleic acid molecule, expression vector, recombinant cell, or drug composition drug used in any embodiment of the present invention can be used to treat or prevent malignant tumors.

[0044] According to an embodiment of the present application, the malignant tumor includes 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.

[0045] It should be understood that within the scope of the present invention, any of the above technical features of the present invention and the technical features specifically described in the following (Implementation Means) can be combined with each other, thereby constituting new or preferred technical means, and will not be repeated here one by one due to space limitations. [Brief explanation of the drawings]

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings.

[0047] [Figure 1] FIG. 1 shows the results of ELISA showing that the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) according to Example 3 of the present invention binds to human and monkey TIGIT. [Figure 2] FIG. 10 shows the ELISA results showing that TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) according to Example 4 of the present invention blocks the binding of TIGIT to CD155. [Figure 3] FIG. 10 shows the results of binding of TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) according to Example 5 of the present invention to 293T-TIGIT cells. [Figure 4] FIG. 10 shows the results of the TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) according to Example 6 of the present invention blocking the binding of CD155-mIgG2a antibody to 293T-TIGIT cells. [Figure 5]FIG. 10 shows the results of TIGIT antibody H401LV2-hlgG1LALA according to Example 7 of the present invention promoting IL-2 secretion from Jurkat T cells. [Figure 6] FIG. 10 shows the results of binding of the TIGIT antibody H401LV2-mlgG2a according to Example 8 of the present invention to CHO-K1-TIGIT cells. [Figure 7] FIG. 10 shows the results of the TIGIT antibody H401LV2-mlgG2a according to Example 9 of the present invention blocking the binding of CD155 to CHO-K1-TIGIT cells. [Figure 8] FIG. 10 shows the results of efficacy of TIGIT antibody H401LV2-mIgG2a in a colorectal cancer MC38 model according to Example 10 of the present invention. [Figure 9] FIG. 10 shows the results of the efficacy of H401LV2-mIgG2aD265A in a lung cancer LLC model according to Example 10 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0048] Hereinafter, the embodiments of the present invention shown in the drawings will be described in detail, and in all the drawings, the same or similar reference numerals indicate the same or similar elements or elements having the same or similar functions. The embodiments described through the following reference drawings are illustrative and are intended to explain the present invention, and should not be understood as limitations on the present invention.

[0049] In the course of describing the present invention, interpretations have been made to the relevant terms in this specification, but these interpretations and explanations are merely for the purpose of facilitating understanding of the means, and are not to be regarded as limitations on the protection means of the present invention.

[0050] In this specification, the terms "comprises" or "comprises" are non-limiting expressions, i.e., include the contents shown in the present invention but do not exclude the contents of other embodiments.

[0051] As used herein, the terms "optionally," "optional," or "optional" generally mean that a described event or circumstance may occur, but does not necessarily occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0052] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art. Abbreviations for amino acid residues refer to the standard three-letter and / or one-letter codes used in the art to represent one of the 20 commonly used L-amino acids.

[0053] In the present invention, unless otherwise specified, the term antigen-binding fragment used is "antibody fragment", and antibody fragment generally refers to an antigen-binding antibody fragment, which may include a portion of an intact antibody, generally the antigen-binding region or variable region, and examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0054] The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence responsible for antigen binding in an antibody, e.g., typically amino acid residues 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and 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, Bethesda, MD. (1991)), and / or "high-variability loops" (e.g., amino acid residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues 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))).

[0055] 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 sequences disclosed herein to obtain variants of the antibody or functional fragment thereof, provided that the antibody activity is not substantially affected (retaining at least 95% activity). These variants are considered to be within the scope of protection provided by the present disclosure. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in the present disclosure have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity as described in the present disclosure can be determined using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. All amino acid sequences referred to in the present disclosure are presented in N- to C-terminal order.

[0056] As used herein, the terms "identity," "homology," or "similarity," when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refer to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as determined by conventional methods, e.g., 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. Alignment of sequences and determination of sequence identity can be achieved by a number of algorithms, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443, the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444, 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, and include, but are 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., FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA, and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0057] As described above, antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies), may comprise 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 with modified glycosylation modes. In some applications, modifications to remove undesired glycosylation sites are useful, or antibodies lacking the lycopersicon gracilis moiety on the oligosaccharide chain are useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications are used to alter complement-dependent cytotoxicity (CDC).

[0058] The term "functional fragment" as used herein refers specifically to antibody fragments, such as Fv, scFv (sc refers to single chain), Fab, F(ab')2, Fab', scFv-Fc fragments, or diabodies, or any fragment whose half-life can be extended by chemical modification or liposome encapsulation, e.g., the addition of poly(alkylene) glycol, such as polyethylene glycol ("PEGylation") (referred to as PEG-, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG pegylated fragments) ("PEG" is polyethylene glycol), and which retain TIGIT-binding activity. Preferably, such functional fragments consist of or comprise a subsequence of the heavy or light chain variable region of the antibody from which they are derived, sufficient to retain the same binding specificity and sufficient affinity for TIGIT as the antibody from which they are derived, preferably at least 100-fold lower, and in a more preferred embodiment, at least 10-fold lower, than the affinity of the antibody from which they are derived. Such functional fragments contain at least 5 amino acids, and preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which it is derived.

[0059] As described above, one aspect of the present invention provides an antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 1, 2, and 3; and They comprise the light chain variable region CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences having at least 80% identity to SEQ ID NOs: 4, 5, and 6, or 4, 5, and 6, respectively.

[0060] In a preferred embodiment of the present invention, an antibody may be humanized to further improve its bioacceptability; i.e., the antibody is a chimeric or humanized antibody. The term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody derived from one species (e.g., mouse) with the constant region of an antibody derived from another species (e.g., human). The term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all amino acid sequences of the constant region and non-CDRs of the variable region (Fv framework region (FR)) of a monoclonal antibody derived from one species (e.g., mouse) with the amino acid sequences of the constant region and non-CDRs of the variable region of an antibody derived from another species (e.g., human). In other words, an antibody in which the constant region of one antibody has been humanized is called a chimeric antibody, and an antibody in which all non-CDR amino acid sequences of the constant region and variable region have been humanized is called a humanized antibody. Humanization methods can be performed using conventional antibody engineering techniques, and therefore will not be described again here.

[0061] In another aspect, the present invention provides a composition, which according to an embodiment of the present invention comprises the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, or recombinant cell.

[0062] The compositions according to the present invention include the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, and recombinant cells. In some embodiments, the compositions include temporally and / or spatially separated combinations of components, as long as they can act cooperatively to achieve the objectives of the present invention. For example, the components included in the compositions can be administered to a subject as a whole or separately. When the components included in the compositions are administered to a subject separately, the components can be administered to the subject simultaneously or sequentially.

[0063] The compositions of the present invention can be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the compositions may further comprise a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present disclosure may be combined with a second therapeutic agent, examples of which include, but are not limited to, other agents that inhibit TIGIT activity (such as other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with TIGIT upstream or downstream signaling.

[0064] Typically, the antibody or antigen-binding fragment thereof is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that causes prevention or alleviation of symptoms associated with the disease being treated, such as a TIGIT-associated disease. The effective amount of the composition administered to a subject will depend on the type and severity of the disease and individual characteristics such as general health, age, sex, weight, and tolerance to drugs, as well as the severity and type of disease, and those skilled in the art will be able to determine the appropriate dose based on these and other factors.

[0065] In a further aspect, the present invention provides a medicament, which, according to an embodiment of the present invention, comprises the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell or composition, and is used to treat malignant tumors.

[0066] The medicament according to the present invention comprises a therapeutic agent and the above-described antibody or antigen-binding fragment thereof that binds to the therapeutic agent. The antibody or antigen-binding fragment thereof binds to the therapeutic agent in a conventional manner.

[0067] In a further aspect, the present invention proposes a kit, according to an embodiment of the present invention, said kit comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention.

[0068] In a further aspect, the present invention proposes the use of the aforementioned antibody or antigen-binding fragment thereof in the preparation of a kit, which, according to an embodiment of the present invention, is used to detect TIGIT.

[0069] A kit for detecting TIGIT in a sample according to the present invention comprises the antibody or its antigen-binding fragment. The sample may be tissue from a patient with a TIGIT-mediated disease (particularly a patient with transplant rejection, autoimmune disease, infectious disease, or cancer, more preferably a patient 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 further comprise a reagent for detecting TIGIT, such as a coating solution.

[0070] The present invention further provides a use of the above-mentioned antibody or antigen-binding fragment thereof in preparing a reagent for detecting TIGIT in a sample. As described above, the sample may be tissue from a patient with a TIGIT-mediated disease, and the details will not be repeated here. The antibody or antigen-binding fragment thereof of the present invention has good affinity with TIGIT and can effectively detect TIGIT in a sample.

[0071] The present invention further provides use of the antibody or antigen-binding fragment thereof in the preparation of a medicament for preventing and / or treating a TIGIT-mediated disease. Preferably, the TIGIT-mediated disease is transplant rejection, autoimmune disease, infectious disease, or cancer. More preferably, the cancer is a TIGIT-expressing cancer. Even more 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, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer. Even more preferably, the infectious disease includes, but is not limited to, HIV virus infection and / or hepatitis B virus infection.

[0072] The present invention further relates to a method for preventing and / or treating a TIGIT-mediated disease (as described above), comprising administering to a patient an effective amount of at least one of the antibodies or antigen-binding fragments thereof, immunoconjugates, and compositions of the present invention, which may be administered orally, intranasally, intradermally, subcutaneously, intramuscularly, intravenously, or intraperitoneally.

[0073] A "patient" or "subject" in relation to the present invention generally refers to a mammal such as a primate and / or rodent, particularly a human or mouse.

[0074] Those skilled in the art can clone a DNA molecule encoding the antibody or antigen-binding fragment thereof of the present invention into a vector (especially an expression vector), transform it into a host cell, and obtain the antibody or antigen-binding fragment thereof by inducible expression. Therefore, the present invention also provides nucleic acid molecules encoding the antibody or antigen-binding fragment thereof and recombinant cells containing the nucleic acid. The nucleic acid is preferably an expression cassette obtained by genetic engineering means.

[0075] The expression vector may refer to a cloning vector or a recombinant vector, which is obtained by operably linking the nucleic acid to a commercially available vector (e.g., a plasmid or viral vector), and commonly used plasmids include pSeTag2, PEE14, pMH3, etc.

[0076] The expression vector of the present invention can contain DNA sequences for the heavy chain variable region, light chain variable region, and / or constant region of the encoded antibody. However, two expression vectors can be constructed, one containing the heavy chain variable region and constant region, and the other containing the light chain variable region and constant region, and transfected together into mammalian cells. In one preferred embodiment, the expression vector contains a promoter, a DNA sequence encoding a secretory signal peptide, and at least one antidrug gene for screening.

[0077] The recombinant cells described herein may be prokaryotic host cells, eukaryotic host cells, or bacteriophages. The prokaryotic host cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic host cells may include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma; insect cells such as grass rotifers; 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 herein are preferably mammalian cells, more preferably BHK cells, CHO cells, NSO cells, or COS cells.

[0078] Nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention, and the corresponding nucleotide sequences can be easily obtained by those skilled in the art based on the amino acid sequences of the heavy and / or light chains.

[0079] As used herein, antibody affinity maturation refers to the normal immune functional state of a living organism. In humoral immunity, the average affinity of antibodies produced in a second response is higher than that of the primary immune response, a phenomenon known as antibody affinity maturation. This functional state of the organism is the result of long-term evolution and continuous adaptation to the external environment and is of great significance for maintaining the organism's defense and autoimmune surveillance. The present invention relates to in vitro antibody affinity maturation technology, specifically, a process in which the CDR amino acids of an antibody are mutated using molecular biology techniques, followed by screening from a mutated antibody library to obtain antibodies with significantly improved affinity.

[0080] Herein, H401LV2 refers to the TIGIT antibody prepared in the present invention.

[0081] As used herein, Ka and k on have the same meaning and refer to the binding constant, and Kd and k off have the same meaning and refer to the dissociation constant.

[0082] As used herein, Jurkat cells refer to suspension cells used to study acute T-cell leukemia, an immortalized human T-lymphocyte lineage that expresses various chemokine receptors that are susceptible to T-cell signaling and viral entry, particularly HIV.

[0083] The embodiments of the present invention shown in the drawings will be described in more detail below. The embodiments described below with reference to the drawings are illustrative and are used to interpret the present invention, but should not be understood as limitations on the present invention.

[0084] In the following examples, the terms "plasmid" and "vector" have the same meaning and can be used interchangeably.

[0085] The parent antibody Hu4A501 used in the following examples refers to the humanized TIGIT antibody developed by this research team, and its sequence is detailed in CN109384846B.

[0086] The tiragolumab analog used is an analog of the anti-human TIGIT antibody tiragolumab developed by Roche, has the same amino acid sequence as tiragolumab, and was purchased from Hyakuei Seibutsu.

[0087] In the following examples, hIgG1 refers to the wild-type human IgG1 constant region, and antibodies of this administration type generally have ADCC (antibody-mediated cytotoxicity) function; hIgG1LALA refers to the human IgG1 constant region with L234A and L235A mutations, which eliminate ADCC function; and hIgG4S228P refers to the human IgG4 constant region with the S228P mutation, and wild-type IgG4 has arm exchange, and the S228P mutation is often performed in antibody engineering to eliminate this effect.

[0088] Example 1: Antibody affinity maturation To improve the suitability of antibodies for drug discovery and obtain high-affinity TIGIT antibodies, the inventors performed affinity maturation (using FASEBA technology, completed by Kingsley Biotechnology) on the developed humanized TIGIT antibody Hu4A501 (CN109384846B). The antigen used for affinity maturation was a recombinant TIGIT extracellular domain Fc fusion protein (TIGIT-Fc) (amino acid sequence shown in SEQ ID NO: 9). The heavy chain variable region and light chain variable region of the obtained matured TIGIT antibody H401LV2 are shown in SEQ ID NO: 7 and 8. QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSS(SEQ ID NO: 7), DIVMTQSPSSMSSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELK(SEQ ID NO:8), MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLELEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 9).

[0089] Example 2: Antibody affinity detection

[0090] The Biacore method is an approved detection method for objectively detecting the affinity and binding kinetics of proteins, and the affinity and binding kinetics of the TIGIT antibody of the present invention were analyzed using Biacore T200.

[0091] TIGIT extracellular domain Fc fusion protein (TIGIT-Fc) was covalently coupled to a CM5 (GE) chip using standard amino acid coupling techniques. A gradient series of TIGIT antibody diluted in PBS buffer was then injected in each cycle, followed by regeneration with 10 mM NaOH solution. Antigen-antibody binding kinetics was monitored for 3 minutes, and dissociation kinetics was monitored for 10 minutes. The data were analyzed using GE's BIAevaluation software using a 1:1 (Langmuir) binding model. The K (k), K (koff), and KD values ​​measured using this method are shown in Table 1.

[0092] [Table 1] 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, and KD represents Kd / Ka, which is the affinity constant.

[0093] Example 3: ELISA binding experiments of TIGIT antibodies

[0094] In accordance with an embodiment of the present invention, the binding properties of TIGIT antibodies are detected using ELISA experiments. TIGIT extracellular domain His-tag fusion proteins are coated onto a 96-well plate, and the binding properties of the antibodies and TIGIT proteins are determined based on the signal intensity after antibody addition. The experimental steps are as follows: 1. Antibody preparation The nucleotide sequences of the heavy and light chains of the above antibodies were respectively stored in pcDNA3.4 vectors (synthesized by Nanjing Jinsirui, Hu4A501 light chain Hu4A501-hK (amino acid sequence shown in SEQ ID NO: 17), Hu4A501 heavy chain H401LV2-hIgG1 (amino acid sequence shown in SEQ ID NO: 18), H401LV2 light chain H401LV2-hK (amino acid sequence shown in SEQ ID NO: 19), H401LV2 heavy chain H401LV2-hIgG1 (amino acid sequence shown in SEQ ID NO: 20), H401LV2-hIgG1LALA (amino acid sequence shown in SEQ ID NO: 21), and H401LV2-hIgG4S228P (amino acid sequence shown in SEQ ID NO: 22). The antibody was prepared by transiently transfecting ExpiCHO-S cells (Gibco, item A29127) with the antibody shown in NO:22. DIVMTQSPSSMSSTSVGDRVTITCRASQDVFTAVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:17) QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:18) DIVMTQSPSSMSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19) QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20) QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:21) QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQG TLLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:22)

[0095] The day before transfection, ExpiCHO-S cells were cultured at a cell density of (3–4) × 10 6 The cells were adjusted to a concentration of 7 × 10 / mL and cultured overnight at 37°C, 8% CO2, and 95 rpm with shaking. On the day of transfection, the cells were cultured at a concentration of 7 × 10 6 ~1×10 7 Grow cells to 6 × 10 / mL and prepare for transfection when viability is greater than 95%. Discard 6 × 10 cells with fresh prewarmed ExpiCHO medium (Gibco, item A2910002). 6The pcDNA3.4 plasmids carrying the heavy and light chains were diluted to 1 mL / mL and transfected into ExpiCHO-S cells with the above heavy and light chain plasmids (1:1 light-heavy chain plasmid ratio) and ExpiFectamine CHO Transfection Reagent (Gibco, Item A29129). The cells were then cultured at 37°C, 8% CO2, and 95 rpm with shaking. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly, and immediately added to the transfected cells. The mixture was then cultured at 32°C, 5% CO2, and 95 rpm with shaking. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and the culture was continued. Cell count and viability were monitored daily. Cells were harvested by centrifugation when cell viability fell below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a Mabselect prism Aprotein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography 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 column was concentrated and replaced with PBS buffer. The purified antibody was identified as having a purity of over 95% by SDS-PAGE, and the results showed that the above antibody was finally obtained.

[0096] 2. ELISA assay to detect the binding characteristics of TIGIT antibody

[0097] 1. Human and cynomolgus monkey TIGIT-His tag fusion proteins (purchased from Acro) were diluted to 2 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and left to stand at 4° C. overnight. 2. Aspirate the PBS buffer from the 96-well plate in step 1, wash the plate six times with PBST buffer (pH 7.2, PBST buffer containing 0.1% Tween 20 by volume), then add 200 μl / well of PBS buffer (containing 10% BSA (bovine serum albumin) by volume) and incubate at 37°C for 2 hours to block. 3. After removing the PBS buffer from step 2, the plate was washed six times with PBST buffer. 100 μl / well of PBST buffer (containing 0.05% volume of BSA) was added to dilute the TIGIT antibody to be measured to 10,000, 2,000, 400, 80, 16, 3.2, 0.64, or 0.128 ng / ml, and the plate was incubated at 37°C for 1 hour. 4. After removing the buffer solution from step 3 and washing the plate six times with PBST, 100 μl / well of HRP (horseradish peroxidase)-labeled human IgG antibody (secondary antibody, purchased from Jackson Lab) was diluted in PBST buffer (containing 0.05% volume of BSA) and incubated at 37°C for 1 hour. 5. Wash the 96-well plate from step 4 six times with PBST buffer, add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and then add 80 μl / well of 4M sulfuric acid to stop the reaction. 6. Read the absorbance at 450 mm of the 96-well plate from step 5 using a microplate reader.

[0098] The results are shown in Figure 1. The TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) prepared in this invention was found to bind to the TIGIT protein, and its binding was stronger than that of the parent antibody Hu4A501 and the tiragolumab analogue (purchased from Hyakuei Bio).

[0099] Example 4: ELISA blocking experiment of TIGIT antibody

[0100] In accordance with an embodiment of the present invention, the binding properties of TIGIT antibodies were detected using ELISA. TIGIT extracellular domain His-tag fusion protein was coated onto a 96-well plate, and antibodies and CD155-biotin were added. The blocking properties of the antibodies against TIGIT / CD155 binding were determined based on the signal intensity. The specific steps are as follows: 1. Human and cynomolgus monkey TIGIT-His tag fusion proteins (purchased from Acro) were diluted to 2 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and left to stand at 4° C. overnight. 2. Aspirate the PBS buffer from the 96-well plate prepared in step 1, wash the plate six times with PBST buffer (pH 7.2, containing 0.1% Tween 20 by volume in PBST buffer), and then add 200 μl / well of 10% BSA (containing 10% by volume of BSA (bovine serum albumin)) and incubate at 37°C for 2 hours to block the plate. 3. Remove the PBS buffer and blocking solution from step 2, wash the plate six times with PBST buffer, add 100 μl / well of PBST buffer (containing 0.05% BSA) / 0.05% BSA to dilute the TIGIT antibody to an appropriate concentration, and incubate at 37°C for 1 hour. 4. After removing the buffer solution from step 3 of the reaction system and washing the plate six times with PBST, 100 μl / well of HRP (horseradish peroxidase)-labeled anti-human IgG secondary antibody (secondary antibody, purchased from Jackson Lab) was diluted in PBST buffer (containing 0.05% BSA) and incubated at 37°C for 1 hour. 5. The 96-well plate from step 4 was washed six times with PBST buffer, 80 μl / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 minutes. Then, 80 μl / well of 4 M sulfuric acid was added to stop the reaction. 6. Read the absorbance at 450 mm of the 96-well plate from step 5 using a microplate reader.

[0101] The results are shown in Figure 2. The TIGIT antibody H401LV2 (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) prepared in the present invention was found to be able to block TIGIT / CD155 binding, and its blocking ability was not weaker than that of the tiragolumab analogue (purchased from Hyakuei Bio).

[0102] Example 5: TIGIT antibody binding 293T-TIGIT experiment

[0103] According to an embodiment of the present invention, the binding characteristics of TIGIT antibody were detected using flow cerebrospinal fluid (FCE) experiments. TIGIT protein (referred to as 293T-TIGIT) was overexpressed in HEK293T cells (ATCC No. CRL-3216), and antibody (which must be supplemented) was added. The binding characteristics of the chimeric antibody and TIGIT were then determined based on the signal intensity. The specific steps are as follows: 1. 5 x 10 HEK293T cells 5 Cells were added to a 6-well plate at 100 cells / well, and DMEM medium without double antibodies was added and cultured overnight. 2. Before transfection, the medium was discarded and 1 ml of fresh double antibody-free DMEM medium was added. pLVX-EF1a-TIGIT-IRES-puro (the TIGIT protein sequence (SEQ ID NO: 11) was inserted between the EcoRI and BamHI enzyme cleavage sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (3 μg total for the three vectors) were added to 200 μl of serum-free DMEM medium in a 2:1:1 ratio. MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFL EVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG(SEQ ID NO:11). 3. 12 μg of polyetherimide (PEI, Polysciences) was added to the medium in step 2. After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate containing HEK293T cells and cultured for 6 hours. 4. The medium in the 6-well plate was discarded, and fresh complete DMEM medium was added and cultured. 48 h after transfection, the supernatant of the cell culture medium was collected and filtered through a 0.45 μm filter (purchased from Millipore) to obtain the viral supernatant. 5. Add all the viral supernatant from step 4 to 1 x 10 4 Polybrene (purchased from Sigma) was added to a 6-well plate containing HEK293T cells at a final concentration of 4 μg / ml and the cells were cultured for 12 hours. 6. The supernatant from step 5 was completely removed and fresh complete DMEM medium was added. The cultured cells were 293T-TIGIT cells. 7. 2 x 10 293T-TIGIT cells in PBS 6 The solution was diluted to 1 / ml and added to 1.5 ml EP tubes at a volume of 100 μl / tube, to which 10 μl / tube of mouse serum was added, followed by blocking at 4° C. for 30 minutes. 8. TIGIT antibody was added to the EP tubes from step 7 at different concentrations (maximum 8 μg / ml, 5-fold dilution, 8 gradients) and incubated at 4°C for 30 minutes. 9. 1 ml of PBS was added to the EP tube from step 8, and the tube was centrifuged at 3500 rpm at 4°C for 5 minutes. The supernatant was completely removed, and the cells were washed once more with PBS. After centrifugation, the supernatant was completely removed, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled mouse anti-human Fc antibody (secondary antibody, purchased from Jackson Lab) was added, and the tube was incubated at 4°C for 30 minutes in the dark. 10. The cells from step 9 were washed with PBS and centrifuged, after which the supernatant was completely removed. The cells were resuspended in 200 μl / tube of PBS buffer and detected using a flow cytometer.

[0104] The results are shown in FIG. 3, and demonstrate that the TIGIT antibody H401LV2 of the present invention (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P) can bind to 293T-TIGIT.

[0105] Example 6: Detection of TIGIT antibody blocking capacity

[0106] TIGIT antibodies block the binding of TIGIT to its ligand CD155 by binding to the extracellular domain of TIGIT. In the present embodiment, the blocking effect of TIGIT antibodies on the binding of CD155 to 293T-TIGIT was detected by flow cytometry. The specific steps are as follows: 1. 293T-TIGIT cells (same as in Example 5) were cultured at 2 x 10 6 The solution was diluted to 1 / ml and added to 1.5 ml EP tubes at a volume of 100 μl / tube, to which 10 μl / tube of mouse serum was added, followed by blocking at 4° C. for 30 minutes. 2. TIGIT antibody and CD155-mIgG2a-Fc (purchased from Acro) diluted 5-fold (total of 10 gradient dilutions) up to 200 μg / ml were added to the EP tube from step 1, and the mixture was incubated at 4°C for 30 minutes. 3. Add 0.1 μg / tube of APC-labeled sheep anti-mouse IgG2a secondary antibody (Biolegend) to the EP tubes from step 2 and incubate at 4°C for 30 minutes. Wash twice with PBS, centrifuge, and completely remove the supernatant. Resuspend the cells in 200 μl / tube of PBS and perform detection using a flow cytometer.

[0107] The results are shown in Figure 4. The TIGIT antibody H401LV2 of the present invention, which has a different constant region (H401LV2-hlgG1, H401LV2-hlgG1LALA, H401LV2-hlgG4S228P), was found to be able to block the binding of CD155-mIgG2a to 293T-TIGIT, and its blocking ability was not weaker than that of the tiragolumab analogue (purchased from Baiying Bio).

[0108] Example 7: Experiment on promotion of Jurkat T cell activation by TIGIT antibody

[0109] Jurkat cells were co-incubated with U-937 tumor cells, and the CD3 and TIGIT antibodies were added to detect the IL-2 content in the supernatant. The activation effect of Jurkat cells was determined based on the intensity of the IL-2 signal after antibody addition. The specific procedure is as follows: 1. OKT3 antibody (purchased from Biolegend) was diluted with PBS to 0.2 μg / ml, added to a 96-well plate, and reacted overnight, and the supernatant was discarded. 2. Incubate 1 x 10 Jurkat cells in Complete 1640 Medium 6 Dilute 1.25 x 10 U-937 cells in complete 1640 medium to 100 µL / ml and add to a 96-well plate in a volume of 100 µL / tube. 6 The cells were diluted to 100 μg / ml and added to a 96-well plate at a volume of 80 μl per tube. TIGIT antibody was diluted to 100 μg / ml in complete 1640 medium and added to the 96-well plate at 20 μl per well. The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours, and IL-2 in the supernatant was detected using a CBA kit (purchased from BD).

[0110] The results are shown in Figure 5 and demonstrate that the affinity-matured TIGIT antibody of the present invention, H401LV2-hlgG1LALA, is more potent at promoting Jurkat T cell activation than the parent antibody, Hu4A501-hlgG1LALA.

[0111] Example 8: TIGIT antibody binding CHO-K1-TIGIT experiment

[0112] In this example, the binding properties of TIGIT antibodies were detected using flow cerebrospinal fluid (FPE) assays. TIGIT protein (referred to as CHO-K1-TIGIT) was overexpressed in CHO-K1 cells (ATCC No. CCL-61). The binding properties of the chimeric antibodies to TIGIT were evaluated based on the signal intensity after antibody addition. 1. Preparation of antibodies: The nucleotide sequences of the heavy and light chains of the above antibodies were respectively placed in the pcDNA3.4 vector (synthesized by Nanjing Jinsirui, including 4.1D3 light chain 4.1D3-mK (amino acid sequence shown in SEQ ID NO: 10), 4.1D3 heavy chain 4.1D3-mIgG2a (amino acid sequence shown in SEQ ID NO: 11), 22G2 light chain 22G2-mK (amino acid sequence shown in SEQ ID NO: 12), 22G2 heavy chain 22G2-mIgG2a (amino acid sequence shown in SEQ ID NO: 13), H401LV2 light chain H401LV2-mK (amino acid sequence shown in SEQ ID NO: 14), and H401LV2 heavy chain H401LV2-mIgG2a (amino acid sequence shown in SEQ ID NO: 15). The antibody was prepared by transient transfection of ExpiCHO-S cells (Gibco, item A29127) with the antibody shown in NO:15. DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEI KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:10), EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:11)、 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLFTFGPGTKVDIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:12)、 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGIYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYYVSGNYYNVDYYFFGVDVWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:13)。 DIVMTQSPSSMSTSVGDRVTITCRASQDVFNQVAWYQQKPGKSPKLLIYSASFRYTGVPDRFSGSGSGTDFTFTISSVQAEDFATYYCQQHYSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:14) QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGT LLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:15)

[0113] The day before transfection, ExpiCHO-S cells were cultured at a cell density of (3–4) × 10 6 The cells were adjusted to a concentration of 7 × 10 / mL and cultured overnight at 37°C, 8% CO2, and 95 rpm with shaking. On the day of transfection, the cells were cultured at a concentration of 7 × 10 6 ~1×10 7 Grow cells to 6 × 10 / mL and prepare for transfection when viability is greater than 95%. Discard 6 × 10 cells in fresh, prewarmed ExpiCHO medium (Gibco, item A2910002). 6The pcDNA3.4 plasmids carrying the heavy and light chains were diluted to 1 mL / mL and transfected into ExpiCHO-S cells with the above heavy and light chain plasmids (1:1 light-heavy chain plasmid ratio) and ExpiFectamine CHO Transfection Reagent (Gibco, Item A29129). The cells were then cultured at 37°C, 8% CO2, and 95 rpm with shaking. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly, and immediately added to the transfected cells. The mixture was then cultured at 32°C, 5% CO2, and 95 rpm with shaking. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and the culture was continued. Cell count and viability were monitored daily. Cells were harvested by centrifugation when cell viability fell below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a Mabselect prism Aprotein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography 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 column was concentrated and replaced with PBS buffer. The purified antibody was identified as having a purity of over 95% by SDS-PAGE, and the results showed that the above antibody was finally obtained.

[0114] 2. Detection of binding characteristics of chimeric antibodies to TIGIT by flow cytometry 1. 5 x 10 HEK293T cells 5 Cells / well were added to a 6-well plate and cultured overnight in DMEM medium without double antibodies. 2. Before transfection, the medium was discarded and 1 ml of fresh double antibody-free DMEM medium was added. The pLVX-EF1a-TIGIT-IRES-puro vector (TIGIT protein inserted between the EcoRI and BamHI enzyme sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (3 μg total of the three vectors) were added to 200 μl of serum-free DMEM medium in a 2:1:1 ratio. 3. 12 μg of polyetherimide (PEI, Polysciences) was added to the medium in step 2, mixed uniformly, and then allowed to stand for 16 minutes. Then, the entire liquid was added to a 6-well plate containing CHO-K1 cells. 4. The 6-well plate from step 3 was cultured for 6 hours, the medium was discarded, and fresh complete DMEM medium was added and cultured. 5. 48 hours after transfection, the supernatant of the cell culture was collected and filtered through a 0.45 μm filter (purchased from Millipore), and the obtained filtrate was the viral supernatant. 6. Add all the viral supernatant to a 1 x 10 4 The mixture was added to a 6-well plate containing 293T cells, and polybrene (purchased from Sigma) was added to a final concentration of 4 μg / ml, followed by incubation for 12 hours. 7. The supernatant was completely removed, and fresh complete DMEM medium was added and cultured. The cultured cells were CHO-K1-TIGIT cells. 8. Dissolve 2 x 10 CHO-K1-TIGIT Cells in PBS 6 The solution was diluted to 1 / ml and added to 1.5 ml EP tubes at a volume of 100 μl / tube, to which 10 μl / tube of mouse serum was added, followed by blocking in a refrigerator at 4° C. for 30 minutes. 9. TIGIT antibody was added at different concentration gradients to the EP tubes from step 8 and incubated in a refrigerator at 4°C for 30 minutes. 1 ml of PBS was added to the EP tubes, which were then centrifuged at 3500 rpm at 4°C for 5 minutes, and the supernatant was completely removed. 10. The cells were again resuspended in PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 100 μl / tube of PBS, to which 1 μl / tube of Alexa-647-labeled mouse anti-human Fc antibody (secondary antibody, purchased from Jackson Lab) was added, and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. 11. The cells were resuspended in 200 μl / tube of PBS and then detected by flow cytometer.

[0115] The results are shown in FIG. 6 and confirm that the TIGIT antibody H401LV2-mlgG2a of the present invention can bind to CHO-K1-TIGIT, and its binding ability is superior to that of the 4.1D3 and 22G2 antibodies.

[0116] Example 9: Detection of TIGIT antibody blocking capacity

[0117] TIGIT antibody blocks the binding of TIGIT to its ligand CD155 by binding to the extracellular domain of TIGIT. The present invention uses flow cytometry to detect the blocking effect of TIGIT antibody on the binding of CD155 to CHO-K1-TIGIT, and the specific steps are as follows: 1. 2 x 10 CHO-K1-TIGIT cells (same as in Example 8) were cultured in PBS. 6 The solution was diluted to 1 / ml and added to 1.5 ml EP tubes at a volume of 100 μl / tube, to which 10 μl / tube of mouse serum was added, followed by blocking at 4° C. for 30 minutes. 2. TIGIT antibody (30, 10, 3, 1, 0.1, 0.01, 0.001 μg / ml) and CD155-mIgG2a-Fc (purchased from Acro) were added and incubated for 30 minutes at 4°C. 0.1 μg / tube of APC-labeled sheep anti-mouse IgG2a (secondary antibody, purchased from Biolegend) was added and incubated for 30 minutes at 4°C. 3. After washing twice with PBS and centrifuging, the supernatant was completely removed. The cells were resuspended in 200 μl of PBS per tube and then detected using a flow cytometer.

[0118] The results are shown in FIG. 7 and confirm that the antibody H401LV2-mlgG2a of the present invention can block the binding of CD155 to CHO-K1-TIGIT, and that the blocking ability is stronger than that of the 4.1D3 antibody.

[0119] Example 10: Effect of H401LV2 antibody on mouse anti-cancer activity

[0120] In vivo efficacy experiments were performed to detect the effect of the affinity-matured TIGIT antibody H401LV2 on the anti-cancer function of hTIGIT transgenic mice. The specific steps were as follows:

[0121] 1. Antibody preparation The antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, Item A29127) with the pcDNA3.4 vector (synthesized by Nanjing Jinsirui; H401LV2 light chain H401LV2-mK (amino acid sequence shown in SEQ ID NO: 14), H401LV2 heavy chain H401LV2-mIgG2a (amino acid sequence shown in SEQ ID NO: 15), and H401LV2-mIgG2aD265A (amino acid sequence shown in SEQ ID NO: 16) containing the nucleotide sequences of the heavy and light chains of the above antibodies. QVQLQESGPGLVKPSQTLSLTCTVTGSSITSDYAWNWIRQFPGKKLEWMGYITYSGRTTYNPSLKSRITISRDTSKNQFSLKLSSVTAADTATYYCARWGLLRRYFDYWGQGT LLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:16)

[0122] The day before transfection, ExpiCHO-S cells were cultured at a cell density of (3–4) × 10 6 The cells were adjusted to a concentration of 7 × 10 / mL and cultured overnight at 37°C, 8% CO2, and 95 rpm with shaking. On the day of transfection, the cells were cultured at a concentration of 7 × 10 6 ~1×10 7 Grow cells to 6 × 10 / mL and prepare for transfection when viability is greater than 95%. Discard 6 × 10 cells in fresh, prewarmed ExpiCHO medium (Gibco, item A2910002). 6The pcDNA3.4 plasmids carrying the heavy and light chains were diluted to 1 mL / mL and transfected into ExpiCHO-S cells with the above heavy and light chain plasmids (1:1 light-heavy chain plasmid ratio) and ExpiFectamine CHO Transfection Reagent (Gibco, Item A29129). The cells were then cultured at 37°C, 8% CO2, and 95 rpm with shaking. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed uniformly, and immediately added to the transfected cells. The mixture was then cultured at 32°C, 5% CO2, and 95 rpm with shaking. Five days after transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed uniformly, and the culture was continued. Cell count and viability were monitored daily. Cells were harvested by centrifugation when cell viability fell below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.45 μm filter, and the Fc domain-containing antibody was captured from the expression supernatant using a Mabselect prism Aprotein A affinity chromatography column (purchased from Suzhou Nawei). After equilibrating the chromatography 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 column was concentrated and replaced with PBS buffer. The purified antibody was identified as having a purity of over 95% by SDS-PAGE, and the results showed that the above antibody was finally obtained. 2. On day 0 of the anti-cancer function test using H401LV2 in hTIGIT transgenic mice, hTIGIT tumor-bearing mice (purchased from Nanfang Model Animals) were cultured at 2 × 10 per mouse. 5 MC38 cells (Figure 8), or 1 x 10 per mouse 6 LLC cells (Figure 9) are injected and the mice are randomly divided into groups. 2. On days 4, 7, 10, and 13, mice are injected intraperitoneally with the affinity-matured antibody at 250 μg per mouse. 3. After the antibody injection, the tumor volume was measured once every three days.

[0123] The results are shown in Figure 8, which demonstrates that H401LV2-mIgG2a with ADCC effector function has a better therapeutic effect in the MC38 colorectal cancer model, and as shown in Figure 9, H401LV2-mIgG2aD265A without ADCC effector function has a better therapeutic effect in the LLC lung cancer model.

[0124] As can be seen from the above experimental results, the high-affinity antibodies obtained in the present invention can bind to TIGIT and activate the immune cell anti-cancer mechanism by blocking the interaction between TIGIT and CD155.

[0125] It should be noted that the terms "first," "second," etc. are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the express number of technical features. Thus, a feature qualified as "first" or "second" may expressly or imply the inclusion of one or more of the feature. Furthermore, in the present description, unless specifically stated otherwise, "plurality" means at least two, e.g., two, three, etc.

[0126] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0127] Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, the technical solutions of the present invention may undergo multiple simple modifications, including combining the respective technical features in other appropriate ways, and these simple modifications and combinations shall also be regarded as the disclosure content of the present invention, and all belong to the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof, heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 1, 2, and 3; and An antibody or antigen-binding fragment thereof, characterized in that it comprises light chain variable region CDR1, CDR2, and CDR3 sequences shown in amino acid sequences having at least 80% identity with SEQ ID NOs: 4, 5, and 6, or 4, 5, and 6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that it comprises a heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 sequence shown in SEQ ID NO: 2, a heavy chain variable region CDR3 sequence shown in SEQ ID NO: 3, a light chain variable region CDR1 sequence shown in SEQ ID NO: 4, a light chain variable region CDR2 sequence shown in SEQ ID NO: 5, and a light chain variable region CDR3 sequence shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof specifically recognizes TIGIT.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that 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 shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a humanized antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain framework region sequence and a light chain framework region sequence, and 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 mouse-derived antibody, a human-derived antibody, a primate-derived antibody, or a variant thereof.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises 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 human-derived antibody, a primate-derived antibody, and a mouse-derived antibody or a mutant thereof.

8. The antibody or antigen-binding fragment of claim 7, wherein the light chain constant region and the heavy chain constant region are both derived from a mouse-derived IgG1 antibody, IgG2a antibody, or a mutant thereof, or a human-derived IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, or a mutant thereof.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is at least one of a single-chain antibody, a multimeric antibody, a CDR-grafted antibody, a Fab antibody, and an Fv antibody.

10. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. An expression vector carrying the nucleic acid molecule of claim 10.

12. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 or 8 to 9, comprising: Introducing the expression vector of claim 11 into a cell; culturing the cells under conditions suitable for protein expression and secretion, and obtaining the antibody or antigen-binding fragment thereof.

13. The method of claim 12, wherein the cell is a eukaryotic cell.

14. The recombinant cell expresses an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and carries a nucleic acid molecule according to claim 10 or an expression vector according to claim 11.

15. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, an expression vector according to claim 11, or a recombinant cell according to claim 14.

16. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5 or 8 to 9, a nucleic acid molecule described in claim 10, an expression vector described in claim 11, a recombinant cell described in claim 14, or a composition described in claim 15 in the preparation of a medicament for treating or preventing malignant tumors.

17. 17. The use of claim 16, wherein the malignant tumor comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

18. A drug for treating a malignant tumor, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the expression vector according to claim 11, the recombinant cell according to claim 14, or the composition according to claim 15.

19. The drug of claim 18, characterized in that the malignant tumor includes at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or 8 to 9 in the preparation of a kit for detecting TIGIT.

21. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

22. A method for treating or preventing a malignant tumor, comprising the step of administering to a subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9, a nucleic acid molecule described in claim 10, an expression vector described in claim 11, a recombinant cell described in claim 14, a composition described in claim 15, or a drug described in claim 18 or 19.

23. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5 or 8 to 9, a nucleic acid molecule described in claim 10, an expression vector described in claim 11, a recombinant cell described in claim 14, a composition described in claim 15, or a drug described in claim 18 or 19 in the treatment or prevention of malignant tumors.

24. 24. The method of claim 22 or the use of claim 23, wherein the malignant tumor comprises at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

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