Anti-TIGIT immune inhibitors and their applications

Abstract

The present invention provides an anti-TIGIT immunoinhibitor and its application, which can bind to the extracellular domain of human TIGIT and can be used to treat diseases such as cancer.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to the technical field of immunology, in particular to immune checkpoint antibodies, their preparation and immunological applications.

[0002] [Background technology] TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a T cell immunoreceptor with immunoglobulin (Ig) and tyrosine inhibitor motif (ITIM) structural domains, and is mainly expressed on activated T cells and NK cells (Yu, X., et al. (2009). "The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells." Nature immunology 10(1):48-57.). TIGIT is also called VSIG9, VSTM3 and WUCAM, indicating that its structure contains one extracellular immunoglobulin structural domain, one type I transmembrane domain and two ITIM motifs. TIGIT is part of a costimulatory network, which is mainly composed of the activating receptor CD226 and the inhibitory receptor TIGIT on T cells, and the ligands CD155 (also called PVR, which is one of the poliomyelitis virus receptor proteins encoded by the PVR gene in humans) and CD112 expressed on the surface of APCs, tumor cells, and infected cells. When TIGIT binds to PVR or CD112, Tyr225 is phosphorylated in the TIGIT cytoplasm, leading to TIGIT binding to the cell self-adaptive growth factor receptor-bound protein 2 (GRB2). GRB2 can recruit SHIP1 to inhibit phosphatidylinositol trikinase (PI3K) and mitogen-activated protein kinase (MAPK) signals. In addition, phosphorylated TIGIT inhibits the recruitment of SHIP1 by beta-arrestin2 and blocks the autoubiquitination of TNF receptor-associated factor 6 (TRAF6) to activate nuclear factor-KB (NF-KB), ultimately terminating the signal transduction pathway. This leads to the inhibition of T cell or NK cell function and the inhibition of cytokine production. PVR is a ligand of TIGIT and also of the CD226 molecule, with the affinity of CD226 for the PVR molecule being 119 nM. Upon binding to CD112 or CD155, Ser329 and Tyr322 in the intracellular structural domain of CD226 are phosphorylated. Phosphorylation of Ser329 promotes the activation of protein kinase C (PKC) and the mutual binding of CD226 to lymphocyte-associated antigen 1 (LFA1). LFA1 is then used for TYN-mediated Tyr322 phosphorylation and CD226-mediated downstream signaling. A series of signaling events ultimately activates T cell or NK cell function and promotes cytokine production. Interaction also occurs between TIGIT and CD226 molecules on the surface of T cells or NK cells, which is reflected in the fact that TIGIT directly interferes with the normal dimer formation of CD226 and disrupts the normal physiological function of CD226. This shows that TIGIT and CD226, like two ends of a scale, ingeniously regulate the body's immune function by co-stimulating and co-inhibiting signal transduction through the fulcrum PVR.

[0003] Studies on the relationship between TIGIT and natural killer (NK) cell dysfunction reveal that TIGIT is associated with NK cell exhaustion in tumor-bearing mice and colon cancer patients. In some tumor-bearing mouse models, blocking TIGIT can prevent NK cell death and promote NK cell-dependent tumor immunity. Furthermore, blocking TIGIT induces effective tumor-specific T cell immunity in an NK cell-dependent manner, improves the therapeutic efficacy of anti-PD-L1 antibodies in tumor rechallenge models, and preserves memory immune competence. This study indicates that TIGIT constitutes a previously unemphasized immune checkpoint on the surface of NK cells and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anti-cancer therapeutic strategy (Zhang, Q., et al. (2018). "Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity." Nature immunology.). TIGIT is highly expressed on the surface of both T cells and NK cells, whereas other immune checkpoints such as PD-1 and CTLA-4 are only expressed on the surface of T cells. As a therapeutic target, TIGIT is certain to have a greater advantage. Therefore, the development of drugs targeting this target may also have bright therapeutic and market prospects.

[0004] Summary of the Invention The present invention provides an anti-TIGIT antibody having high affinity for human TIGIT protein. In some embodiments, the anti-TIGIT antibody of the present invention is an isolated anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody of the present invention can effectively block the binding of TIGIT to PVR, and can also effectively activate lymphocytes to release cytokines. In some embodiments, the anti-TIGIT antibody of the present invention has high affinity for human TIGIT and appropriate ADCC (antibody-dependent cellular cytotoxicity) activity. In some embodiments, the anti-TIGIT antibody of the present invention can be used for therapeutic purposes to treat various cancers and infectious diseases, and can also be used for diagnosis and prognosis.

[0005] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to an Immunoglobulin and T Cell Immunoreceptor with Tyrosine-Based Inhibitory Motif (TIGIT) protein and comprises one, two, three, four, five or all of the following sequences: (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having a single substitution, deletion or insertion therein; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22 or an amino acid sequence having a single substitution, deletion or insertion therein; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having a single substitution, deletion or insertion therein; (d) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having a single substitution, deletion or insertion therein; (e) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 or 43, or an amino acid sequence having a single substitution, deletion or insertion therein; and (f) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27 or an amino acid sequence having a single substitution, deletion or insertion therein.

[0006] In some embodiments, the invention provides an isolated antibody or fragment thereof, wherein the antibody or fragment thereof specifically binds to a T cell immunoreceptor with Immunoglobulin and Immunoreceptor Tyrosine-Based Inhibitory Motif (TIGIT) protein and comprises: (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having a single substitution, deletion or insertion therein; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22 or an amino acid sequence having a single substitution, deletion or insertion therein; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having a single substitution, deletion or insertion therein; (d) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having a single substitution, deletion or insertion therein; (e) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:43, or an amino acid sequence having a single substitution, deletion or insertion therein; and (f) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27 or an amino acid sequence having a single substitution, deletion or insertion therein.

[0007] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof, which specifically binds to T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motifs (TIGIT) protein, and the antibody or fragment thereof comprises one, two, three, four, five or all of a VH CDR1 set forth in SEQ ID NO:21, a VH CDR2 set forth in SEQ ID NO:22, a VH CDR3 set forth in SEQ ID NO:23, a VL CDR1 set forth in SEQ ID NO:25, a VL CDR2 set forth in SEQ ID NO:26 or 43, and a VL CDR3 set forth in SEQ ID NO:27.

[0008] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof, which specifically binds to T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motifs (TIGIT) protein, and the antibody or fragment thereof comprises a VH CDR1 set forth in SEQ ID NO:21, a VH CDR2 set forth in SEQ ID NO:22, a VH CDR3 set forth in SEQ ID NO:23, a VL CDR1 set forth in SEQ ID NO:25, a VL CDR2 set forth in SEQ ID NO:26 or 43, and a VL CDR3 set forth in SEQ ID NO:27.

[0009] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof specifically binds to T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motifs (TIGIT) protein, and the antibody or fragment thereof comprises a VH CDR1 set forth in SEQ ID NO:21, a VH CDR2 set forth in SEQ ID NO:22, a VH CDR3 set forth in SEQ ID NO:23, a VL CDR1 set forth in SEQ ID NO:25, a VL CDR2 set forth in SEQ ID NO:26, and a VL CDR3 set forth in SEQ ID NO:27.

[0010] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof, which specifically binds to T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motifs (TIGIT) protein, and the antibody or fragment thereof comprises a VH CDR1 set forth in SEQ ID NO:21, a VH CDR2 set forth in SEQ ID NO:22, a VH CDR3 set forth in SEQ ID NO:23, a VL CDR1 set forth in SEQ ID NO:25, a VL CDR2 set forth in SEQ ID NO:43, and a VL CDR3 set forth in SEQ ID NO:27.

[0011] In some embodiments, the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain is of the kappa or lambda type. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the light chain constant region is a kappa chain constant region. In some embodiments, the light chain variable region is a kappa or lambda chain variable region. In some embodiments, the light chain variable region is a kappa chain variable region. In some embodiments, the antibody or fragment thereof is of one of the following isotypes: IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antibody or fragment thereof is a humanized antibody.

[0012] In some embodiments, the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region heavy chain has an E amino acid at position 356 (EU numbering). In some embodiments, the human Fc region heavy chain has an M amino acid at position 386 (EU numbering). In some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:28 or 29. In some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO:30.

[0013] In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 33, 35, 37, 39, and 41, or a peptide chain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 33, 35, 37, 39, and 41. In some embodiments, the antibody or fragment thereof comprises a light chain variable region, the light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 34, 36, 38, 40, and 42, or a peptide chain having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 34, 36, 38, 40, and 42.

[0014] In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO:35 and a light chain variable region whose amino acid sequence is set forth in SEQ ID NO:36.

[0015] In some embodiments, the antibody or fragment thereof can effectively block the binding of TIGIT to PVR. In some embodiments, the antibody or fragment thereof can activate lymphocytes to release cytokines.

[0016] In some embodiments, the antibody or fragment thereof has an affinity value for human TIGIT, K D In some embodiments, the antibody or fragment thereof has an affinity value K D is less than 5 nM.

[0017] In some embodiments, the antibody or fragment thereof has ADCC activity. In some embodiments, the antibody or fragment thereof is conjugated to fucose. In some embodiments, the antibody or fragment thereof is not conjugated to fucose.

[0018] In another aspect, the present invention further describes a bispecific antibody, characterized in that it comprises an antibody fragment according to the invention and a second antigen-binding fragment having specificity for a molecule on an immune cell, including PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR and CD47.

[0019] In another aspect, the present invention further provides a composition comprising the antibody or fragment thereof disclosed herein, the composition further comprising a pharma- ceutically acceptable carrier. In some embodiments, 50% or less of the antibodies or fragments thereof in the composition are bound to fucose. In some embodiments, 30% or less of the antibodies or fragments thereof in the composition are bound to fucose. In some embodiments, 20% or less of the antibodies or fragments thereof in the composition are bound to fucose. In some embodiments, 10% or less of the antibodies or fragments thereof in the composition are bound to fucose. In some embodiments, 5% or less of the antibodies or fragments thereof in the composition are bound to fucose. In some embodiments, 1% or less of the antibodies or fragments thereof in the composition are bound to fucose.

[0020] In another aspect, the present invention further provides a polynucleotide capable of encoding the antibody or fragment thereof, in some embodiments, the sequence of the polynucleotide encoding the antibody heavy chain is set forth in SEQ ID NO:46, and in some embodiments, the sequence of the polynucleotide encoding the antibody light chain is set forth in SEQ ID NO:47.

[0021] In another embodiment, the present invention further provides a cell comprising one or more polynucleotides encoding the antibody or fragment thereof disclosed in the present invention.

[0022] In another aspect, the present invention further provides a method for preparing an antibody or fragment thereof, comprising culturing a cell comprising a polynucleotide encoding the antibody or fragment thereof to express the antibody or fragment thereof, in some embodiments, the cell is a CHO cell, a HEK293 cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a mouse L cell.

[0023] In another aspect, the present invention further provides methods of treatment and uses. In some embodiments, the present invention provides a method of treating cancer or an infectious disease in a patient in need thereof, comprising administering to the patient an effective dose of an antibody or fragment thereof disclosed herein. In one embodiment, the cancer is a solid tumor. In some embodiments, the cancer is bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer. In some embodiments, the cancer may be bladder cancer, liver cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, and small cell lung cancer. In some embodiments, the method further comprises administering to the patient a second cancer therapeutic agent. In some embodiments, the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

[0024] In some embodiments, the present invention provides a method of treating cancer or an infectious disease in a patient in need thereof, comprising: (a) treating cells ex vivo with an antibody or fragment thereof disclosed herein; and (b) administering the treated cells to the patient. In some embodiments, the method further comprises isolating the cells from the individual prior to step (a). In some embodiments, the cells are isolated from within the patient. In some embodiments, the cells are isolated from a donor individual different from the patient. In some embodiments, the cells are T cells, non-limiting examples of which include tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0025] In some embodiments, the present invention further provides the use of an anti-TIGIT antibody or fragment thereof in the preparation of a medicament for treating cancer or an infectious disease in a patient in need thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer. In some embodiments, the method further comprises administering to the patient a second cancer therapeutic agent. In some embodiments, the infectious disease is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

[0026] In some embodiments, the present invention further provides an application of the cells in the preparation of a medicament for treating cancer or an infectious disease, the application comprising: (a) treating the cells with an antibody or a fragment thereof in vitro; and (b) administering the treated cells to a patient. In some embodiments, the method further comprises isolating the cells from an individual prior to step (a). In some embodiments, the cells are isolated from within the patient. In some embodiments, the cells are isolated from a donor individual different from the patient. In some embodiments, the cells are T cells. In some embodiments, the T cells are tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0027] In another aspect, the present invention further provides a diagnostic method and use. In some embodiments, the present invention provides a method for detecting the expression of TIGIT in a sample, comprising contacting the sample with an antibody or a fragment thereof under certain conditions, thereby binding the antibody or a fragment thereof to TIGIT, and detecting the binding, which is the expression of TIGIT in the sample. In some embodiments, the sample comprises a tumor cell, a tumor tissue, an infected tissue, or a blood sample.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly explain the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings that need to be used in the embodiments. Of course, the drawings below are merely some of the embodiments described in the present application.

[0029] [Table 1]

[0030] JPEG0007628111000002.jpg223169

[0031] JPEG0007628111000003.jpg233169

[0032] JPEG0007628111000004.jpg83169

[0033] [Mode for carrying out the invention] definition It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus the terms "a" (or "one"), "one or more" and "at least one" may be used interchangeably herein.

[0034] An "amino acid" is an organic compound that contains both an amino group and a carboxyl group, such as α-amino acids, and can be encoded by a nucleic acid, either as such or in the form of a precursor. A single amino acid is encoded by a nucleic acid that consists of three nucleotides (a so-called codon or base triplet). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called the "degeneracy of the genetic code." Amino acids include natural and unnatural amino acids. Natural amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0035] "Conservative amino acid substitution" means that one amino acid residue is replaced with another amino acid residue that contains a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein. Examples of classes of amino acids that contain side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) acidic side chains: aspartic acid and glutamic acid.

[0036] In the present invention, the term "polypeptide" is intended to cover a singular "polypeptide" and a plurality of "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly connected through amide bonds (also called peptide bonds). The term "polypeptide" refers to any single or multiple chains consisting of two or more amino acids and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of or interchangeably with any one of the above terms. The term "polypeptide" also refers to products modified after expression of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting or blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide can be derived from a natural organism or produced by recombinant technology, but is not necessarily obtained by translation from a specified nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0037] The term "isolated" as used herein with respect to antibodies, cells, and nucleic acids, e.g., "isolated" DNA or RNA, refers to a molecule that is isolated from other DNA or RNA, respectively, present in the natural origin of the large molecule. As used herein, the term "isolated" refers to a nucleic acid or peptide that is essentially free of cellular material, viral material, or cell culture medium, when produced by recombinant DNA technology, or chemical precursors or other chemicals, when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not found in the natural state, and are not found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are isolated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides.

[0038] As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide refers to a polypeptide or polynucleotide that does not occur in nature, a non-limiting example being one that can be combined to produce a polynucleotide or polypeptide that does not normally occur.

[0039] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The term "unrelated" or "non-homologous" sequence indicates a sequence having less than 40% identity, e.g., less than 25% identity, with one of the sequences disclosed in the present invention.

[0040] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence, which means that the percentage of bases (or amino acids) are the same in the two sequences being compared when the sequences are aligned. The alignment and percentage of homology or sequence identity can be determined by software programs known in the art, such as those described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those polynucleotides that have the percentages of homology specified above and that encode polypeptides having the same or similar biological activity.

[0041] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity with the nucleotide sequence of a nucleic acid or its complement. A double-stranded nucleic acid homolog is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology with itself or its complementary sequence. Meanwhile, a nucleic acid homolog can hybridize with a nucleic acid or its complement. Similarly, an "equivalent polypeptide" is a polypeptide having a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some embodiments, compared to a reference polypeptide or polynucleotide, an equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some embodiments, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0042] Hybridization reactions can be carried out under conditions of different "stringency". Low stringency hybridization reactions are usually carried out at about 40° C. in about 10×SSC or a solution of the same ionic strength / temperature. Medium stringency hybridization reactions are usually carried out at about 50° C. in about 6×SSC, and high stringency hybridization reactions are usually carried out at about 60° C. in about 1×SSC. Hybridization reactions may be carried out under "physiological conditions", which are well known to those skilled in the art. Non-limiting examples of physiological conditions include temperature, ionic strength, pH and MgCl2 typically present in cells. 2+ Refers to the concentration.

[0043] A polynucleotide consists of a specific sequence of four nucleotide bases, namely adenine (A), cytosine (C), guanine (G) and thymine (T), where thymine is replaced by uracil (U) when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" refers to the letters of a polynucleotide molecule, which can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one form of a gene or part thereof, and a part of a gene that has at least two different forms (i.e., two different nucleotide sequences) is called a "polymorphic region of a gene." A polymorphic region may be a mononucleotide, but has different identities in different alleles.

[0044] The terms "polynucleotide" and "oligonucleotide" may be used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of polynucleotides are genes or gene fragments (e.g., probes, primers, EST or SAGE labels), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, carriers, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the structure of the nucleotides, if any, may be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide moieties. After polymerization, the polynucleotide may be further modified, such as by conjugation with a labeling moiety. The term refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide embodiment of the present disclosure includes the double-stranded form and each of the two complementarable single-stranded forms that make up the known or predicted double-stranded form.

[0045] The term "code," when applied to a polynucleotide, refers to a polynucleotide that "encodes" a polypeptide, which, in its natural state or when manipulated by methods known to those of skill in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complementary sequence of such a nucleic acid, from which the coding sequence can be derived.

[0046] In the present invention, an "antibody" or an "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be an intact antibody and any antigen-binding fragment thereof or a single chain thereof. Thus, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule in the molecule that has the biological activity of binding to an antigen. Examples include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the heavy chain variable region (VH) or light chain variable region (VL), the heavy chain constant region (CH) or light chain constant region (CL), the frame (FR) region or any portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR region of the light chain (LCDR) and the CDR region of the heavy chain (HCDR).

[0047] In the present invention, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as an F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, enantiomers, and bivalent antibodies. The term "antibody fragment" further includes any synthetic or genetically engineered protein that binds to a specific antigen to form a complex and performs the action of an antibody.

[0048] A "single chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, these regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker may be glycine-rich to improve flexibility or serine- or threonine-rich to improve solubility, and may be connected to the N-terminus of the VH and the C-terminus of the VL, or vice versa. The protein has the constant regions removed and the linker introduced, but retains the specificity of the original immunoglobulin. ScFv molecules known in the art are described, for example, in U.S. Pat. No. 5,892,019.

[0049] The term "antibody" includes a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that the types of heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and further include several subclasses (e.g., γ1-γ4). Depending on the nature of the chain, the "type" of the antibody is determined as IgG, IgM, IgA, IgD, or IgE, respectively. For example, immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, have already been fully characterized, and the functional specificities conferred by them are also known. Those skilled in the art will easily conceive various modifications of these types and isotypes, and it will be clear that all of them are within the scope of the claims disclosed in the present invention, and that all immunoglobulin types are within the scope of the claims disclosed in the present invention. For IgG, a standard immunoglobulin molecule includes two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000 to 70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, with the light chains starting at the mouth of the "Y" and continuing to surround the heavy chains through their variable regions.

[0050] The antibodies, antigen-binding fragments or derivatives disclosed in the present invention may be polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab' and F(ab') 2 Immunoglobulin or antibody molecules disclosed herein include, but are not limited to, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, Fvs connected by disulfide bonds (sdFv), fragments comprising the VK or VH structural domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, the anti-Id antibodies of the LIGHT antibody disclosed herein). The immunoglobulin or antibody molecules disclosed herein may be of any immunoglobulin type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0051] Light chains can be divided into kappa or lambda (κ, λ). Each heavy chain can be bound to a κ or λ light chain. Generally, when an immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the light and heavy chains are bound via a covalent bond, and the "tails" of the two heavy chains are bound via a covalent disulfide bond or a non-covalent bond. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The κ light chain variable region of an immunoglobulin is Vκ, and the λ light chain variable region of an immunoglobulin is Vλ.

[0052] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used according to function. In this regard, it should be understood that the variable regions of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant regions of the light (CL) and heavy (CH1, CH2 or CH3) chains confer important biological properties such as secretion, placental transport, Fc receptor binding, complement fixation, etc. According to convention, the numbering of the constant regions increases the further they are from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL structural domains comprise the carboxy termini of the heavy and light chains, respectively.

[0053] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the combination of the VL structural domain and the VH structural domain or a subset of the complementarity determining regions (CDRs) of the antibody forms the variable region that defines a three-dimensional antigen-binding site. The quaternary structure of the antibody forms the antigen-binding site at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) in each of the VH and VL chains. In some cases, for example, immunoglobulin molecules derived from some camelids or immunoglobulin molecules modified based on camelid immunoglobulins, the complete immunoglobulin molecule may be composed of only heavy chains without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0054] In naturally occurring antibodies, when the antibody assumes its three-dimensional configuration in an aqueous environment, the six "complementarity determining regions" or "CDRs" in each antigen-binding domain are specifically positioned to form a short, non-contiguous amino acid sequence of the antigen-binding domain. The remaining amino acids in the antigen-binding domain, called "framework" regions, show little inter-molecular variability. Most of the framework regions adopt a β-fold conformation, forming a ring structure to which the CDRs are attached, or in some cases, forming part of the β-fold structure. Thus, the framework regions form a stent, positioning the CDRs in the correct orientation in inter-chain non-covalent interactions. The antigen-binding domain formed by the CDRs at specific positions defines a surface complementary to the epitope in the immunoreactive antigen, which promotes non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, one of skill in the art can identify the amino acids that comprise the CDRs and framework regions by commonly used definitions (e.g., "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0055] The CDRs defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. However, either definition of the CDRs of an antibody or antigen-binding fragment thereof is within the scope of the definition and term used in the present invention. The exact residue numbers that comprise a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR by the amino acid sequence of the variable region of the antibody.

[0056] Kabat et al. also defined a numbering system that can be applied to any antibody variable region sequence. One of skill in the art can, of course, apply the "Kabat numbering" system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983).

[0057] The Kabat numbering system describes the CDR regions as follows: HCDR1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. HCDR2 begins at about amino acid 15 after HCDR1 ends, includes about 16-19 amino acid residues, and ends at the next arginine or lysine residue. HCDR3 begins at about amino acid 33 after HCDR2 ends, includes 3-25 amino acids, and ends with the sequence WGXG, where X represents any amino acid. LCDR1 begins at about amino acid 24 (i.e., after the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. LCDR2 begins at about amino acid 16 after LCDR1 ends, and includes about 7 residues. LCDR3 begins about the 33rd residue after LCDR2 ends (ie, after the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG, where X represents any amino acid.

[0058] The antibodies disclosed herein may be derived from any animal, including birds and mammals. Preferably, the antibodies are of human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken origin. In other embodiments, the variable region may be of condricthoid origin (e.g., from sharks).

[0059] A "light chain-heavy chain pair" is an assembly of a light chain and a heavy chain that can form a dimer through disulfide bonds between the CL structural domain of the light chain and the CH1 structural domain of the heavy chain.

[0060] As mentioned above, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin types are known. In the present invention, the term "VH structural domain" includes the amino-terminal variable structural domain of an immunoglobulin heavy chain, and the term "CH1 structural domain" includes the first (most amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 structural domain is adjacent to the VH structural domain, and the CH1 structural domain is connected to the amino-terminus of the hinge region of the immunoglobulin heavy chain molecule.

[0061] In the present invention, the term "CH2 structural domain" includes the portion of the heavy chain molecule of an antibody that extends from about residue 244 to residue 360 ​​when the conventional numbering scheme is used (residues 244-360, Kabat numbering system; residues 231-340, EU numbering system; see Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983)). The CH2 structural domain is unique because it is not tightly paired with other structural domains, but rather has two N-linked branched carbohydrate chains inserted between the two CH2 structural domains of an intact native IgG molecule. The CH3 structural domain is also described in the literature as extending from the CH2 structural domain to the C-terminus of the IgG molecule and containing about 108 residues.

[0062] In the present invention, the term "hinge region" includes the portion of the heavy chain that connects the CH1 structural domain and the CH2 structural domain. The hinge region contains about 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct structural domains: the upper, middle, and lower hinge structural domains (Roux et al., J. Immunol 161:4083 (1998)).

[0063] In the present invention, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. Cysteine ​​contains a thiol group that can form a disulfide bond with a second thiol group or crosslink. In most naturally occurring IgG molecules, the CH1 and CL regions are connected by a disulfide bond, and the two heavy chains are connected by two disulfide bonds at corresponding positions 239 and 242 in the Kabat numbering system (positions 226 or 229, EU numbering system).

[0064] In the present invention, the term "chimeric antibody" is taken to refer to any antibody whose immunoreactive region or site is obtained or derived from a first species and whose constant region (which in the present invention may be complete, partial or modified) is derived from a second species. In some embodiments, the target binding region or site is of non-human (e.g., murine or primate) origin and the constant region is of human origin.

[0065] "Specific binding" generally refers to an antibody binding to an antigen epitope via its antigen-binding domain, and the binding requires complementarity between the antigen-binding domain and the epitope. According to this definition, when an antibody binds to an epitope via its antigen-binding domain more easily than when it binds to a random or unrelated epitope, it is said to "specifically bind" to the epitope. The term "specific" is used in the present invention to qualify the relative affinity of a particular antibody to bind to a particular epitope.

[0066] In the present invention, the term "treatment" refers to therapeutic treatment and prophylactic or preventive therapeutic measures, aimed at preventing or reducing (reducing) the progression of adverse physiological changes or disorders, such as cancer. Beneficial or desired clinical results include, but are not limited to, alleviation or elimination of symptoms, reduction in disease severity, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation, and alleviation or elimination (whether partial or total), whether detectable or not. "Treatment" further means prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those suffering from a disease or disorder, and those susceptible to a disease or disorder, or those in need of prevention of said disease or disorder.

[0067] "Subject" or "individual" or "animal" or "patient" or "mammal" generally refers to any subject in need of diagnosis, prognosis or treatment, particularly a mammalian subject. Mammalian subjects include humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc.

[0068] As used herein, the collocations, e.g., "patient in need of treatment" or "subject in need of treatment", include subjects, e.g., mammalian subjects, who would benefit from the administration of the antibodies or compositions disclosed herein during detection and diagnostic procedures and / or treatment.

[0069] Anti-TIGIT antibody The present invention provides anti-TIGIT antibodies with high affinity to human TIGIT protein. The tested antibodies exhibit effective binding and inhibitory activity and can be used for therapeutic and diagnostic purposes. For example, as shown in the Examples, these antibodies can effectively block the binding of TIGIT to PVR and can also effectively activate lymphocytes to release cytokines. These antibodies have high affinity to human TIGIT and suitable ADCC activity.

[0070] Thus, the present invention provides an anti-TIGIT antibody or a fragment thereof capable of specifically binding to a TIGIT protein.

[0071] In some embodiments, the present invention provides antibodies comprising heavy and light chain variable domains of CDR regions as shown in Table 2.1. As shown in the experiments in the Examples, antibodies comprising these CDR regions, whether murine or humanized, have effective binding and inhibitory activity against TIGIT.

[0072] In some embodiments, the anti-TIGIT antibody disclosed in the present invention comprises a VH set forth in SEQ ID NO: 20, 33, 35, 37, 39, and / or 41, or a sequence having 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, respectively, and a VL set forth in SEQ ID NO: 24, 34, 36, 38, 40, and / or 42, or a sequence having 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, respectively. In some embodiments, the VH having 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto as a whole retains the CDRs (amino acid sequences of SEQ ID NO: 21-23, 25-27, 43, or a sequence having a single substitution, deletion or insertion therein). In one example, the VH has the amino acid sequence set forth in SEQ ID NO:35 and the VL has the amino acid sequence set forth in SEQ ID NO:36.

[0073] In some embodiments, the anti-TIGIT antibody disclosed in the present invention comprises a human Fc region. The heavy and light chain constant region sequences may have, for example, SEQ ID NO:28 (or 29) and 30. Compared to SEQ ID NO:28, SEQ ID NO:29 has a D to E substitution at amino acid position 239 (position 356 in EU numbering, position 377 in Kabat numbering) and an L to M substitution at amino acid position 241 (position 358 in EU numbering, position 381 in Kabat numbering).

[0074] In some embodiments, the anti-TIGTI antibody disclosed in the present invention has a heavy chain sequence shown in SEQ ID NO:44 and a light chain sequence shown in SEQ ID NO:45.

[0075] One skilled in the art should further understand that the antibodies disclosed in the present invention can be modified such that after modification their amino acid sequence differs from that of the naturally occurring binding polypeptide from which the antibody was derived. For example, a polypeptide or amino acid sequence derived from the same designated protein may be similar to the starting sequence, e.g., have a certain percentage of identity with the starting sequence, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence.

[0076] In some embodiments, the antibody comprises an amino acid sequence and one or more groups not normally associated with an antibody. Representative modifications are described in more detail below. For example, the antibodies disclosed herein may comprise flexible linker sequences or may be modified to add functional groups (e.g., PEG, drugs, toxins, or labels).

[0077] In some embodiments, the antibody or fragment thereof is not conjugated to fucose. In some embodiments, the antibody or fragment thereof has at most three (or at most two, one) amino acid residues modified with fucose. In some embodiments, a formulation comprising the antibody or fragment thereof has at most less than 0.01%, 0.1%, 1%, 2%, 3%, 4% or 5% of protein molecules modified with fucose. In some embodiments, antibodies with reduced or eliminated fucose modification have stronger ADCC and are suitable for some therapeutic uses.

[0078] The antibodies, variants or derivatives disclosed in the present invention include derivatives that are modified, i.e., modified by the covalent attachment of any type of molecule to the antibody, where the covalent attachment does not prevent the antibody from binding to the epitope. For example, including but not limited to the following examples, the antibodies may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by conventional techniques, including but not limited to specific chemical degradation, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

[0079] In some embodiments, the antibody can be conjugated to a therapeutic agent, a drug precursor, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a drug, or PEG.

[0080] Antibodies can be conjugated or fused to therapeutic agents, which may include detectable labels, such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photosensitizers and diagnostic agents, cytotoxic agents such as drugs or toxins, ultrasound enhancing agents, non-radioactive labels and compositions thereof, and other such reagents known in the art.

[0081] The antibody is detectably labeled by coupling it to a chemiluminescent compound, and the presence of the chemiluminescent-tagged antibody is determined by detecting the emission of light emitted during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridine salts, and oxalate esters.

[0082] bifunctional molecule TIGIT is both an immune receptor and a tumor antigen. As a tumor antigen targeting molecule, an antibody or antigen-binding fragment that specifically binds to TIGIT can be combined with another antigen-binding fragment that has immune cell specificity to produce a bispecific antibody.

[0083] In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells. Target molecules on immune cells include, for example, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1, CTLA-4, LAG-3 (also called CD223), CD28, CD122, 4-1BB (also called CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also called CD272), killer cell immunoglobulin-like receptors (KIR), and CD47. Specific examples of bispecific antibodies include, but are not limited to, TIGIT / PD-1, TIGIT / LAG3, and TIGIT / CD47. As shown in the Examples, the TIGIT antibody and PD-L1 antibody of the present invention have a synergistic effect.

[0084] In some embodiments, an antibody or antigen-binding fragment that specifically binds to TIGIT can be combined with another antigen-binding fragment that has tumor antigen specificity to produce a bispecific antibody. A "tumor antigen" is an antigenic substance produced by tumor cells, i.e., that elicits an immune response in the host. Tumor antigens can be used to identify tumor cells and can be used as candidate targets for cancer therapy. Normal proteins in the body are not antigenic, but rather, certain proteins are produced or overexpressed during tumor development, so they are "exogenous" to the body. These proteins may include normal proteins that are well sequestered by the immune system, proteins that are normally produced in very small amounts, proteins that are normally produced only during some stages of development, or proteins whose structure has been modified by mutation.

[0085] A large number of tumor antigens have been discovered in the art, and new tumor antigens can be readily identified through screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucoproteins, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, intrinsic protein, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin.

[0086] In some embodiments, the monovalent unit has specificity for a protein that is overexpressed in tumor cells compared to corresponding non-tumor cells. As used herein, "corresponding non-tumor cells" refers to non-tumor cells of the same cell type as the origin of the tumor cells. It should be noted that such proteins are not necessarily distinct from tumor antigens, and non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectal, breast, lung, pancreatic and gastrointestinal cancers; neuromodulatory protein receptors (HER-2, neu or c-erbB-2), which are consistently overexpressed in breast, ovarian, colon, lung, prostate and cervical cancers; and epidermal growth factor receptor (EGFR), which is highly expressed in a range of solid tumors, including breast, head and neck, non-small cell lung and prostate cancers. GFR), asialoglycoprotein receptor, transferrin receptor, serpin complex receptor expressed in hepatocytes, fibroblast growth factor receptor (FGFR) overexpressed in pancreatic ductal adenocarcinoma cells, vascular endothelial growth factor receptor (VEGFR) for antiangiogenic gene therapy, folate receptor selectively overexpressed in 90% of non-mucinous ovarian cancers, cell surface glycocalyx, carbohydrate receptor, and polymerized immunoglobulin receptor, which primarily delivers genes to respiratory epithelial cells and has potential appeal for the treatment of cystic fibrosis, etc. Non-limiting examples of bispecifics include TIGIT / EGFR, TIGIT / Her2, TIGIT / CD33, TIGIT / CD133, TIGIT / CEA, and TIGIT / VEGF.

[0087] The present invention further discloses different formats of bispecific antibodies. In some embodiments, the anti-TIGIT fragment and the other fragment are each selected from a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0088] The present invention further discloses bifunctional molecules other than antibodies or antigen-binding fragments. As tumor antigen targeting molecules, antibodies or antigen-binding fragments that specifically bind to TIGIT according to the present invention can be selectively linked to immunocytokines or ligands via peptide linkers. The linked immunocytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine the effect of immune checkpoint blockade with local immune regulation at the tumor site.

[0089] Polynucleotides encoding antibodies and methods for preparing antibodies The present invention further discloses isolated polynucleotides or nucleic acid molecules encoding the antibodies and antigen-binding fragments or derivatives thereof according to the present invention. The polynucleotides disclosed in the present invention can encode the entire heavy and light chain variable regions of said antibodies or antigen-binding fragments or derivatives. The polynucleotides disclosed in the present invention can also encode a portion of the heavy and light chain variable regions of said antibodies or antigen-binding fragments or derivatives.

[0090] In some embodiments, the sequence of a polynucleotide encoding the anti-TIGIT antibody h10D8OF is shown in SEQ ID NO:46 and 47, of which the sequence SEQ ID NO:46 can encode a heavy chain that produces the anti-TIGIT antibody h10D8OF, and the sequence SEQ ID NO:47 can encode a light chain that produces the anti-TIGIT antibody h10D8OF.

[0091] The sequence of SEQ ID NO:46 is as follows:

[0092] [Table 2]

[0093] The sequence of SEQ ID NO:47 is as follows:

[0094] [Table 3]

[0095] In some embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention are prepared and produced by hybridoma technology. For example, the hybridoma method prepares monoclonal antibodies such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent such that lymphocytes produce or are capable of producing antibodies that specifically bind to the immunizing agent.

[0096] The immunizing agent typically includes a protein antigen, a fragment thereof, or a fusion protein thereof. Typically, peripheral blood lymphocytes are used when human cells are desired, and splenic lymphocytes or lymph node cells are used when non-human mammalian sources are desired. In some embodiments, splenic lymphocytes are used. The lymphocytes are then fused with an immortalized cell line to form hybridoma cells by a suitable fusing agent, such as polyethylene glycol (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is typically a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Typically, a rat or mouse myeloma cell line is used. In some embodiments, the fusion is performed using splenic lymphocytes and mouse myeloma cells. The hybridoma cells can be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, when the parent cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymine ("HAT medium"), which prevents the growth of HGPRT-deficient cells. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is measured by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, for therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0097] Once the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Media suitable for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium.

[0098] In some embodiments, the monoclonal antibodies secreted from the subclones can be isolated or purified by conventional techniques, such as, for example, protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0099] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies described herein can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells described herein are preferred sources of such DNA. DNA encoding the antibodies described herein can also be designed and synthesized by conventional methods according to the antibody sequence. The isolated or synthesized DNA can be placed into an expression carrier and then transfected into host cells, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, monkey COS cells, PER.NS0 cells, SP2 / 0, YB2 / 0, and myeloma cells, that do not otherwise produce immunoglobulins, to obtain monoclonal antibodies synthesized in the recombinant host cells.

[0100] In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is prepared and produced by hybridoma technology, in which a mouse is immunized with a fusion protein containing the TIGIT extracellular domain, and after immunization, splenic lymphocytes of the mouse are taken and fused with myeloma cells, and hybridomas are selected by the fusion protein containing the TIGIT extracellular domain, and the positive hybridomas are subjected to limiting dilution and further subcloned, and the binding ability of the hybridoma strain to the fusion protein containing the TIGIT extracellular domain is re-identified, thereby preparing and producing the anti-TIGIT antibody. In some embodiments, the fusion protein containing the TIGIT extracellular domain is TIGIT-Fc. In some embodiments, the mouse is a 6- to 8-week-old female Balb / c mouse.

[0101] Methods for preparing antibodies are known in the art and described herein. In some embodiments, both the variable and constant regions of the antibodies or antigen-binding fragments thereof disclosed herein are fully human. Fully human antibodies can be prepared by techniques disclosed in the art and described herein. For example, fully human antibodies to a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but without using its endogenous locus. Exemplary techniques for preparing such antibodies are described in U.S. Pat. Nos. 6,150,584, 6,458,592, and 6,420,140, ​​the entire contents of which are incorporated herein by reference.

[0102] In some embodiments, the antibodies prepared do not provoke an adverse immune response in the animal to be treated, such as a human. In some embodiments, the antibodies disclosed herein or their antigen-binding fragments or derivatives are modified by art-recognized techniques to reduce their immunogenicity. For example, the antibodies may be humanized, primatized, deimmunized, or prepared as chimeric antibodies. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, and retain or essentially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved in a variety of ways, including (a) grafting the entire non-human variable region onto a human constant region to produce a chimeric antibody, (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) onto a human frame and constant region, with or without retaining important frame residues, or (c) grafting the entire non-human variable region, but "masking" them by replacing surface residues with human-like moieties.

[0103] Deimmunization can also be used to reduce the immunogenicity of antibodies. "Deimmunization" includes modifying T cell epitopes by altering the antibody (see, for example, International Application Publication Nos. WO / 9852976A1 and WO / 0034317A2). For example, the variable heavy and variable light chain sequences from a starting antibody are analyzed to generate a human T cell epitope "spectrum" showing the location of the epitopes from each V region relative to the complementarity determining regions (CDRs) and other important residues in the sequence. Selectable amino acid substitutions with low risk of altering the final antibody activity are identified by analyzing single T cell epitopes from the T cell epitope map. A set of selectable variable heavy and variable light chain sequences containing combinations of amino acid substitutions are designed, and these sequences are then incorporated into a set of binding polypeptides. Typically, 12-24 antibody variants are generated and their binding ability and / or function are detected. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into an expression vehicle, and the plasmids are subsequently introduced into cell lines to produce the complete antibody. The antibodies are then compared in appropriate biochemical and biological experiments to identify the optimal variant.

[0104] The binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0105] Examples of techniques that can be used to make single chain Fvs (scFvs) and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498, as well as Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993), and Skerra et al., Science 240:1038-1040 (1988), and other known methods.

[0106] For some applications, including the use of antibodies in the human body and in vitro detection experiments, chimeric, humanized or fully human antibodies are preferably used. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as an antibody having a mouse monoclonal antibody variable region and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986), Gillies et al., J. Immunol. Methods 125:191-202 (1989) and U.S. Patents 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.

[0107] A humanized antibody is an antibody molecule derived from a non-human antibody capable of binding to a target antigen, said antibody having one or more complementarity determining regions (CDRs) of the non-human and a combination of frame regions from a human immunoglobulin molecule. Usually, some frame residues in the human frame regions are replaced with corresponding residues from the CDR donor antibody, preferably residues that can improve antigen binding. These frame replacements can be identified by methods known in the art, for example, simulating the interaction of CDRs and frame residues to identify frame residues that play an important role in antigen binding, and comparing sequences to identify unusual frame residues at specific positions. (See Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), the entire contents of which are incorporated herein by reference). For example, antibodies can be humanized by a variety of techniques known in the art, such as CDR grafting (EP 239,400, PCT publication WO91 / 09967, US Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106, EP 519,596, Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska.et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain rearrangement (US Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0108] For the treatment of human patients, fully human antibodies are particularly desirable. Fully human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, the entire contents of each of which are incorporated herein by reference.

[0109] Human antibodies can also be produced in transgenic mice, which are incapable of expressing functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, a complex of human heavy and light chain immunoglobulin genes can be randomly introduced into mouse embryonic stem cells or introduced by homologous recombination. Alternatively, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional by introducing human immunoglobulin loci, either individually or simultaneously, by homologous recombination. In particular, homozygous deletion of the JH region can prevent the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastulae to produce chimeric mice. The chimeric mice are then bred to produce homozygous progeny expressing human antibodies. The transgenic mice are immunized by conventional methods with a selected antigen, e.g., all or a portion of a desired polypeptide target. Monoclonal antibodies against target antigens can be obtained from immunized transgenic mice by conventional hybridoma technology. Human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation, followed by categorical conversion and somatic mutation. Thus, such technology can produce IgG, IgA, IgM and IgE antibodies that can be used for treatment. For a review of the technology for producing such fully human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of the technology for producing fully human antibodies and human monoclonal antibodies and processes for producing such antibodies, see PCT publications WO 98 / 24893, WO 96 / 34096, WO 96 / 33735, and U.S. Patents Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598, the entire contents of which are incorporated herein by reference.Moreover, companies such as Abgenix (Freemont, Calif.) and GenPharm (San Jose, Calif.) can provide fully human antibodies directed against a selected antigen using technology similar to that described above.

[0110] Fully human antibodies that recognize a selective epitope can also be produced by a technique called "guided selection," in which a selected non-human monoclonal antibody, such as a mouse antibody, guides the selection of a fully human antibody that recognizes the same epitope (Jespers et al., Bio / Technology 72:899-903 (1988) and U.S. Patent 5,565,332, the entire contents of which are incorporated herein by reference).

[0111] In some alternative embodiments, DNA encoding the desired monoclonal antibody can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding mouse antibody heavy and light chains). The isolated and subcloned hybridoma cells provide a suitable source of such DNA. Once isolated, the DNA can be placed in an expression carrier and transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic, as described herein) can be used to clone constant and variable region sequences for preparing antibodies, as described, for example, in Newman et al. and U.S. Pat. No. 5,658,570, published Jan. 25, 1995, the entire contents of which are incorporated herein by reference. In practice, this requires the extraction of RNA from the selected cells, transformation into cDNA, and then amplification by PCR techniques utilizing Ig-specific primers. Suitable probes adapted for this purpose may be found in U.S. Patent 5,658,570. As described in more detail below, transformed cells expressing the desired antibodies may be cultured in relatively large quantities to meet the clinical and commercial demand for immunoglobulins.

[0112] Furthermore, humanized, non-fully human antibodies can be constructed by inserting one or more CDRs of the antibody or antigen-binding fragment of the invention into a frame region, for example, into a human frame region, using standard recombinant DNA techniques. The frame region may be a naturally occurring or common frame region, but is preferably a human frame region (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a range of human frame regions). Preferably, the polynucleotide produced by combining the frame region and the CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions are made in the frame region, with amino acid substitutions that can improve binding of the antibody to its antigen being preferred. Also, antibody molecules lacking one or more interchain disulfide bonds can be produced in this manner by substituting or deleting cysteine ​​residues in one or more variable regions involved in the formation of interchain disulfide bonds. Other alterations made to the polynucleotide within the skill of the art are encompassed by the present invention.

[0113] Furthermore, chimeric antibody technology (Morrison et al., Proc. Natl. Acad. Sci. USA: 851-855 (1984); Neuberger et al., Nature 372: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)) relies on splicing a gene with appropriate antigen specificity from a mouse anti-molecule and a gene from an available human anti-molecule with appropriate biological activity. In the present invention, a chimeric antibody is a molecule whose different portions are derived from different animal species, such as, for example, a chimeric antibody comprising a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

[0114] Additionally, Newman, Biotechnology 10:1455-1460 (1992) discloses another efficient method for producing recombinant antibodies, which, among other things, can produce primate antibodies containing monkey variable regions and human constant region sequences, the entire contents of which are incorporated herein by reference. Additionally, this technology is also referenced in commonly assigned U.S. Patents 5,658,570, 5,693,780, and 5,756,096, the entire contents of which are incorporated herein by reference.

[0115] Antibodies can be prepared by conventional recombinant DNA techniques. Antibody-producing carriers, cell lines, etc. can be selected, constructed, and cultured by techniques known to those skilled in the art. All of these techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including any supplementary content, are incorporated herein by reference in their entirety.

[0116] In some embodiments, DNA encoding the antibodies described herein can be designed and synthesized according to the antibody amino acid sequence according to conventional methods, placed in an expression carrier, transfected into host cells, and cultured in a medium to produce monoclonal antibodies. In some embodiments, the expression antibody carrier includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites used for RNA splicing on either side of the insertion sequence. Efficient transcription can be obtained from the early and late promoters of SV40, long terminal repeats from retroviruses, such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, but other cellular promoters, such as the actin promoter, may also be applied. Suitable expression vehicles may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian host cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0117] In some embodiments, the inserted gene fragment must contain a selection marker, common selection markers include selection genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, etc., so that successfully transfected cells can be selected and isolated. The constructed plasmid is transfected into host cells lacking the above genes and selectively cultured in a medium, and the successfully transfected cells are mass-produced to produce the desired target protein.

[0118] Furthermore, mutations may be introduced into the nucleotide sequence encoding the antibody according to the invention by standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Preferably, the variants (including derivatives) encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the referenced heavy chain variable region HCDR1, HCDR2, HCDR3 and light chain variable region LCDR1, LCDR2 or LCDR3. Alternatively, mutations may be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and mutants that retain activity may be identified by screening for biological activity of the resulting mutants.

[0119] Cancer Treatment In accordance with the present invention, the antibodies, antigen-binding fragments or derivatives of the invention can be used in a number of therapeutic and diagnostic methods.

[0120] The present invention further describes antibody-based therapy, in which an antibody according to the invention is administered to a patient, such as an animal, mammal or human, to treat one or more disorders or conditions as described herein. Therapeutic compounds of the invention include, but are not limited to, the antibodies according to the invention (including antigen-binding fragments and derivatives according to the invention) and nucleic acids or polynucleotides encoding the antibodies according to the invention (including antigen-binding fragments and derivatives according to the invention).

[0121] The antibodies described in the present invention may be used to treat or inhibit cancer. TIGIT can be overexpressed in tumor cells. Tumor-derived TIGIT can limit anti-tumor T cell immunity by binding to PD-1 in immune cells. The results of using small molecule inhibitors or monoclonal antibodies targeting TIGIT in mouse tumor models reveal that TIGIT-targeted therapy is an important alternative and practical method to effectively control tumor growth. As confirmed in the examples, anti-TIGIT antibodies can improve the survival rate of cancer patients by activating adaptive immune response mechanisms.

[0122] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. In one embodiment, the method entails administering to the patient an effective dose of an antibody described in the present invention. In some embodiments, at least one cancer cell (e.g., matrix cell) in the patient's body expresses, overexpresses, or inducibly expresses TIGIT. For example, inducible expression of TIGIT can be achieved by administration of a tumor vaccine or radiation therapy.

[0123] Tumors expressing TIGIT protein include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colon cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, and small cell lung cancer. Thus, the antibodies disclosed in the present invention can be used to treat any one or more of such cancers.

[0124] The present invention further provides cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used to contact with (or, alternatively, engineer to express) the anti-TIGIT antibody described in the present invention. Through such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer described herein. The cells (e.g., T cells) include, but are not limited to, the following types, such as tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, and combinations thereof.

[0125] In some embodiments, the cells are isolated from the cancer patient's own body. In some embodiments, the cells are provided by a donor or a cell bank. When the cells are isolated from the cancer patient, adverse immune responses can be minimized.

[0126] The antibodies or antigen-binding fragments or derivatives thereof according to the invention may be used to treat, prevent, diagnose and / or predict other diseases or conditions associated with increased cell viability, including, but not limited to, the progression or metastasis of malignant tumors and / or related disease disorders, including leukemia {including acute leukemias [e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)] and chronic leukemias [e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia]}, polycythemia vera, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, for example, fibrosarcoma, myxosarcoma, liposarcoma, sarcoma ... Liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma Cancer, including, but not limited to, renal cell carcinoma, hepatic carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, sarcomas and carcinomas such as neuroblastoma and retinoblastoma.

[0127] Combination therapy In some alternative embodiments, the compositions of the present invention are administered in combination with an antitumor, antiviral, antibacterial, or antibiotic or antifungal agent, any of which are known in the art can be administered in the compositions of the present invention.

[0128] In some other embodiments, the compositions of the present invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present invention include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin D), antiestrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouridine, interferon alpha-2b, glutamic acid, mithramycin, mercaptopurine, and 6-thioguanine), cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, methylhydrazine, mitomycin, busulfan, cisplatin, and vincristine sulfate), hormones, and the like. These include, but are not limited to, steroids (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone), nitrogen mustard derivatives (e.g., melphalan, chlorambucil, dichloromethyldiethylamine (mechlorethamine), and thiotepa), steroids and combinations thereof (e.g., betamethasone sodium phosphate), and other compounds (e.g., dacarbazine, aspartic acid, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0129] In some embodiments, the compositions of the invention are administered in combination with cytokines, including, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, CD40, CD40L, and TNF-α.

[0130] In some embodiments, the compositions of the invention are administered in combination with other therapeutic or prophylactic regimens, such as radiation therapy.

[0131] The present invention further provides combination therapy comprising using one or more anti-TIGIT antibodies of the present invention and a second anti-cancer agent (chemotherapeutic agent). Examples of chemotherapeutic agents include, but are not limited to, therapeutic antibodies, including immunotherapeutic agents, that are applied in the treatment of patients. Some examples of therapeutic antibodies are simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectuximab, and cefotaximab. bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab AB), conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab uximab), ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab,Glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab b), ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab,These include teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and 3F8. Rituximab can be used to treat indolent B-cell cancers, including marginal zone lymphoma, WM, CLL, and small lymphocytic lymphoma. The combination of rituximab with chemotherapy drugs is highly effective.

[0132] In some embodiments, the compounds and compositions described herein can be used or combined with one or more other therapeutic agents. The one or more therapeutic agents may be selected from the group consisting of Abl inhibitors, activated CDC kinase (ACK), adenosine A2B receptor (A2B), apoptosis signal-regulating kinase (ASK), Auroa kinase, Bruton's tyrosine kinase (BTK), BET-bromo structural domain (BRD) such as BRD4, c-Kit, c-Met, CDK activating kinase (CAK), calmodulin-dependent protein kinase (CaMK), cyclin-dependent kinase (CDK), casein kinase (CK), discoidin domain receptor (DDR), epidermal growth factor receptor (EGFR), focal adhesion kinase (FAK), Flt-3, FYN, glycogen synthase kinase (GSK), HCK, histone deacetylase (HDAC), IKK such as IKKβε, isocitrate dehydrogenase (IDH) such as IDH1, Janus kinase (JAK), KDR, lymphocyte-specific protein tyrosine kinase (LCK), lysyl oxidase protein, lysyl oxidase-like protein (LOXL), LYN, matrix metalloproteases (MMPs), MEK, mitogen-activated protein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derived growth factor (PDGF), phosphorylase kinase (PK), polo-like kinase (PLK), phosphatidylinositol 3-kinase (PI3K), protein kinases (PK) such as protein kinase A, B and / or C, PYK, spleen tyrosine kinase (SYK), serine / threonine kinase TPL2, serine / threonine kinase STK, signal transduction and transcription (STAT), SRC, serine / threonine protein kinases (TBK) such as TBK1, TIE, tyrosine kinase (TK), vascular endothelial growth factor receptor (VEGFR), or any combination of the above.

[0133] In some embodiments, the anti-TIGIT antibody of the present invention can be used together with an immune checkpoint inhibitor. An immune checkpoint is a type of molecule in the immune system, and can be an upregulation signal (costimulatory molecule) or a downregulation signal (co-inhibitory molecule). Many cancers protect themselves from immune system attacks by inhibiting T cell signals with agonists of co-inhibitory molecules or antagonists of co-stimulatory molecules. An immune checkpoint agonist or antagonist can contribute to blocking such a protective mechanism. The immune checkpoint agonist or antagonist may target any one or more of the following checkpoint molecules: PD-L1, PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 / OX40L, CD40 / CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, or BTLA (also known as CD272). In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody is one or more of Pembrolizumab, Nivolumab, Toripalimab, Sintilimab, Tislelizumab, Camrelizumab, and Genolimzumab. In some embodiments, the anti-PD-1 antibody is Pembrolizumab. In some embodiments, the anti-PD-L1 antibody is one or more of Atezolizumab, Avelumab, Durvalumab, Envafolimab, and Cosibelimab. In some embodiments, the anti-PD-L1 antibody is Atezolizumab.

[0134] For any of the above combination therapies, the anti-TIGIT antibody can be administered simultaneously or separately with the other anti-cancer agent. When administered alone, the anti-TIGIT antibody can be administered before or after the administration of the other anti-cancer agent.

[0135] Treating infection As confirmed in the experimental examples, the antibodies of the invention are capable of activating the immune response and are therefore used in the treatment of infectious diseases.

[0136] Infection is due to the invasion of pathogenic agents into living tissues, their reproduction, and the host tissues' response to the organism and their toxins. Infection can be caused by infectious agents such as, for example, viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as mites, acarids, fleas and lice, fungi such as vermis, and other macroparasites such as tapeworms and other helminths. In some embodiments, the infectious agent is a bacterium, such as a gram-negative bacterium. In some embodiments, the infectious agent is a virus, such as a DNA virus, an RNA virus, and a retrovirus. Non-limiting examples of viruses include adenovirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpes virus type 8, HIV, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, poliomyelitis virus, rabies virus, respiratory syncytial virus, rubella virus, and varicella-zoster virus.

[0137] The antibodies of the present invention can also be used to treat infectious diseases caused by microorganisms, or to target and bind to immune cells to kill the microorganism, thereby achieving the goal of eliminating the microorganism. In some embodiments, the microorganism is a virus, including RNA and DNA viruses, a gram-positive bacterium, a gram-negative bacterium, a protozoan, or a fungus. Table 4 provides non-limiting examples of infectious diseases and associated microorganisms.

[0138] Treatment method The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody and antigen-binding fragment or derivative thereof used, the patient's age and weight, general health, sex and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are within the skill of a medical professional to determine. The dosage will further depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage to be used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0139] Methods of administration of antibodies and antigen-binding fragments thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, spinal epidural injection, and oral administration. The antibodies or compositions may be administered by any convenient route, such as infusion or push injection, may be absorbed through epithelial or cutaneous mucosa (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Thus, pharmaceutical compositions containing the antibodies of the present invention may be administered orally, rectally, parenterally, intracerebrally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drop, or transdermal patch), buccally, or by oral or nasal spray.

[0140] The term "enterogastric" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0141] The mode of administration may be systemic or local. Additionally, the antibody of the present invention may need to be introduced into the central nervous system by any suitable route, including intraventricular and intrathecal injection, which may be assisted by being connected to something like a liquid capsule (which may be an Ommaya liquid capsule) via an intraventricular catheter. Pulmonary administration may also be used, for example, by using an inhaler or nebulizer, and by using an atomized formulation.

[0142] It may be necessary to administer the antibody polypeptides or compositions of the invention locally to the area to be treated, which may be achieved by, but is not limited to, local infusion during surgery, topical application following surgery in conjunction with, for example, a wound dressing, by injection, by catheter, by suppository, or by implants, which may be porous, non-porous, or gel-like materials, including membranes (e.g., silicone rubber membranes) or fibers. Preferably, when administering the proteins (including antibodies) of the invention, care must be taken to use materials that do not absorb the proteins.

[0143] In some embodiments, the compositions of the invention comprise a nucleic acid or polynucleotide encoding a protein, which is constructed as part of a suitable nucleic acid expression carrier, whereby the nucleic acid is administered internally to promote expression of the protein encoded thereby, and the partial carrier can be administered to become an intracellular part, for example, by using a retroviral carrier (see U.S. Pat. No. 4,980,286), or by direct injection, or by using biolistic techniques (e.g., gene gun; Biolistic, Dupont), or by coating with liposomes or cell surface receptors or transfection reagents, or by administration in conjunction with a homeobox peptide that has entered the cell nucleus as known (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for use in expression.

[0144] Dosages of the antibodies of the invention can be determined by standard clinical techniques that effectively treat, inhibit, and prevent inflammatory, immune, or malignant disorders, disorders, or diseases. In addition, in vitro experiments can optionally be used to help determine optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or disease, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be estimated from dose-response curves derived from in vitro or animal model test systems.

[0145] In some embodiments, the dose of the antibody of the present invention administered to a patient is typically 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 20 mg / kg, or 1 mg / kg to 10 mg / kg. Typically, due to immune responses to exogenous polypeptides, human antibodies have a longer half-life in the human body than antibodies of other species. Thus, low doses of human antibodies and low administration frequency are typically feasible. Furthermore, the administration dose and frequency of the antibody of the present invention can be reduced by improving the uptake and tissue penetration ability (e.g., entering the brain) of the antibody through modifications such as liposomization.

[0146] Typically, in vitro assays for treating infectious or malignant diseases, disorders or conditions include administering an antibody, antigen-binding fragment or derivative according to the present invention, and in vivo assaying for the desired therapeutic or prophylactic activity in an acceptable animal model, and finally administering to humans. Suitable animal models (including transgenic animals) are known to those skilled in the art. For example, in vitro assays for confirming therapeutic use of an antibody or antigen-binding fragment according to the present invention include the effect of the antibody or antigen-binding fragment on a cell line or a patient tissue sample. The effect of the antibody or antigen-binding fragment on a cell line and / or tissue sample can be detected by techniques known to those skilled in the art, such as those disclosed elsewhere in the present invention. In vitro assay experiments that can be used to determine whether to administer a specific antibody or antigen-binding fragment according to the present invention include in vitro cell culture experiments, in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound, and the effect of such compound on the tissue sample is observed.

[0147] A variety of delivery systems are known and can be used to administer an antibody of the invention, or a polynucleotide encoding an antibody of the invention, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of the nucleic acid as part of a retrovirus or other carrier, etc.

[0148] Diagnostic methods Overexpression of TIGIT has been observed in some tumor samples, and patients with cells that overexpress TIGIT may respond to treatment with the anti-TIGIT antibody of the present invention. Therefore, the antibody of the present invention can also be used for diagnosis and prognosis.

[0149] Preferably, the sample containing cells can be obtained from a patient, which may be a cancer patient or a patient to be diagnosed. The cells can be cells from a tumor tissue or tumor block, a blood sample, a urine sample or any sample from a patient. After optional pretreatment of the sample, the sample can be incubated with the antibody of the invention under conditions that allow the interaction of the antibody with the TIGIT protein that may be present in the sample. The anti-TIGIT antibody can be used to detect the presence of TIGIT protein in the sample by methods such as ELISA.

[0150] The presence (any content or concentration) of TIGIT protein in a sample can be used to diagnose cancer, which may be an indication that a patient should be administered antibody therapy, or that a patient has responded (or not responded) to a cancer treatment. Prognostic methods can be detected once, twice or more times at a particular stage when cancer treatment is initiated, to indicate the progression of the treatment.

[0151] composition The present invention further provides a pharmaceutical composition. Such a composition comprises an effective dose of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0152] In a specific embodiment, the term "pharmaceutical acceptable" refers to a material listed in the Pharmacopoeias for animal, and particularly human, use. Furthermore, a "pharmaceutical acceptable carrier" is typically any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation additive.

[0153] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier administered for treatment. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions, aqueous glucose solutions, and glycerin solutions may be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and the like. If necessary, the composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, and phosphates. Antibacterial agents such as benzyl alcohol and methyl p-hydroxybenzoate, antioxidants such as ascorbic acid and sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity such as sodium chloride and dextrose are also foreseeable. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions may be prepared as suppositories with conventional adhesives and carriers such as triglycerides. Oral formulations may contain standard carriers such as drug levels of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions contain a clinically effective dose of the antibody, preferably in purified form, combined with an appropriate amount of carrier to provide a dosage form suitable for the patient. The formulation should be adapted to the mode of administration. The parent formulation may be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0154] In some embodiments, the composition is prepared by a conventional process into a pharmaceutical composition suitable for intravenous injection into humans. Typically, compositions for intravenous administration are solutions in sterile or other water-permeable buffers. If necessary, the composition may further include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Generally, the active ingredient is provided alone or in admixture in unit dose form, for example in a sealed container (e.g., an ampoule or sachet) that indicates the amount of active ingredient in the form of a dry lyophilized powder or water-free concentrate. When the composition is administered by infusion, the composition may be packaged separately in an infusion bottle containing sterile medical water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be used so that the active ingredients can be mixed before administration.

[0155] The compounds of the present invention may be prepared in a neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0156] In the following examples, unless otherwise specified, the reagents and equipment used are common in the art and can be commercially obtained, and the methods used are common in the art. Those skilled in the art can certainly carry out the examples and obtain corresponding results based on the contents described in the examples.

[0157] Some cell construction: References: Yu, X., et al. (2009). "The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells." Nature immunology 10(1):48-57. and Neumann, F., et al. (1995). "Human ribosomal protein L7 inhibits cell-free translation in reticulocyte lysates and affects the expression of nuclear proteins upon stable transfection into Jurkat T-lymphoma cells." Nucleic acids research 23(2):195-202. Jurkat cells, sAPC cells, and CTI-Jurkat cells overexpressing human TIGIT are constructed using the techniques described above.

[0158] Construction of sAPC cells: The nucleotide sequence of the heavy chain variable region encoding mouse anti-human CD3 single chain antibody (SEQ ID NO.51), the linker (SEQ ID NO.52), the nucleotide sequence of the light chain variable region of mouse anti-human CD3 single chain antibody (SEQ ID NO.53) and the nucleotide sequence encoding PVR (SEQ ID NO.54) are transfected into CHO-K1 cells (ATCC, CCL-61) to obtain a cell line sAPC that stably expresses human PVR and mouse anti-human CD3 single chain antibody in the cell membrane.

[0159] Construction of CTI-Jurkat cells: Human full-length TIGIT gene (SEQ ID NO. 55), human full-length CD226 gene (SEQ ID NO. 56), IL-2 response element (SEQ ID NO. 57), and luciferase reporter gene plasmid pNFκB-TA-luc (Biyuntian, D2207) were transfected into Jurkat cell lines (ATCC, Clone E6-1, TIB-152, TM) to obtain a cell line, CTI-Jurkat, capable of stably expressing human TIGIT and human CD226 molecules.

[0160] Construction of Jurkat cells overexpressing human TIGIT: Human full-length TIGIT gene (SEQ ID NO. 55) was transfected into Jurkat cell lines (ATCC, Clone E6-1, TIB-152 TM ) to obtain a cell line, Jurkat-hTIGIT 2E6, capable of stably expressing human TIGIT.

[0161] [Table 4]

[0162] JPEG0007628111000008.jpg157169

[0163] Working Example Example 1 Expression and purification of PVR-Fc and TIGIT-Fc fusion proteins The amino acid residue sequence of PVR extracellular domain (SEQ ID NO:1) was sequence-optimized according to human codon preference characteristics, and the nucleotide sequence SEQ ID NO:2 was obtained after optimization. An enzyme cleavage site was added and fused with human IgG1 constant domain sequence (SEQ ID NO:28, the nucleotide sequence of which is shown in the underlined part of SEQ ID NO:46) via a linker (linker, SEQ ID NO:6 and 7). As shown in the schematic diagram (Figure 1), the fused sequence was inserted into pCDNA3.1+ carrier (Invitrogen, V790-20), and then 293F cells were transiently transfected. The cultured cell supernatant was purified by Protein A affinity chromatography, and the purified fusion protein was named PVR-Fc.

[0164] The amino acid residue sequence of the extracellular domain of TIGIT (SEQ ID NO:3) was sequence-optimized according to human codon preference characteristics, and the nucleotide sequence SEQ ID NO:48 was obtained after optimization. An enzyme cleavage site was added and fused with the human IgG1 constant domain sequence (SEQ ID NO:28, the nucleotide sequence of which is shown in the underlined part of SEQ ID NO:46) via a linker (linker, SEQ ID NO:6 and 7). As shown in the schematic diagram (Figure 2), the fused sequence was inserted into pCDNA3.1+ carrier, and then 293F cells were transiently transfected. The cultured cell supernatant was purified by Protein A affinity chromatography, and the purified fusion protein was named TIGIT-Fc.

[0165] [Table 5]

[0166] JPEG0007628111000010.jpg240169

[0167] JPEG0007628111000011.jpg188169

[0168] Example 2 Production of mouse anti-human TIGIT antibodies Female Balb / c mice aged 6 to 8 weeks (Guangdong Medical Experimental Animal Center) were immunized with TIGIT-Fc described in Example 1, which is a protein formed by fusing the extracellular domain of human TIGIT with human IgG-Fc. The mice were immunized again 2 to 3 weeks after the first immunization, and then boosted about 3 weeks later. The spleens were then removed to prepare splenic lymphocytes, and the splenic lymphocytes were fused with myeloma cells (Sp2 / 0 cells, Cell Bank of the Committee for Culture Collection of Typical Samples, Chinese Academy of Sciences, product number TCM18). Hybridomas were selected based on whether they could effectively bind to TIGIT-Fc. The positive hybridomas were further subcloned by limiting dilution, and then their binding ability to TIGIT-Fc was identified again, and the binding ability of TIGIT-Fc to PVR-Fc was blocked. Finally, six monoclonal hybridomas were obtained, one of which showed good expression of various characteristics, and the monoclonal hybridoma of this strain was 10D8.

[0169] According to the method described in the literature (Bradbury, A. (2010). Cloning Hybridoma cDNA by RACE. Antibody Engineering. R. Kontermann and S. Dubel. Berlin, Heidelberg, Springer Berlin Heidelberg: 15-20), specific primers were synthesized and the hybridoma cell antibody target gene was cloned. The hybridoma sequencing process is as follows: 1) The hybridoma cells were resuscitated, placed in a T25 cell culture flask, the medium was RPMI1640 (10% FBS), and passaged for two generations. 2) Five million hybridoma cells were taken and RNA was extracted with EasyPure RNA kit (Beijing Full-Scale Gold Biotechnology Co., Ltd., product number: ER101). 3) The extracted RNA was used as a template to synthesize cDNA with a reverse transcription reagent kit (Beijing Full-Scale Gold Biotechnology Co., Ltd., product number: AT321). 4) Then, the cDNA was used as a template to amplify the target band of the antibody sequence with the synthesized specific primer. FastPfu (Beijing Full-Scale Gold Biotechnology Co., Ltd., product number: AP221) was used to prevent mutations during amplification. 5) The amplified heavy and light chain target fragments were cloned into blunt-end clone carriers (Beijing Full-Scale Gold Biotechnology Co., Ltd., product number: CB111) respectively, and then spread onto plates respectively. 6) After the clones grew, five heavy chain clones and five light chain clones were selected respectively and sent to Sankyo Biotechnology (Shanghai) Co., Ltd. for sequencing. 7) The sequencing results were analyzed by analysis programs such as NCBI blast tool and NCBI IGBlast Tool to find the heavy chain variable region sequence and light chain variable region sequence of the antibody. 8) The variable region sequence of the mouse anti-human TIGIT antibody was codon optimized based on mouse codon selection, and the sequence was synthesized.The synthesized variable region sequence was fused with the mouse IgG2a heavy chain constant region frame (SEQ ID NO:31, SEQ ID NO:49) and the mouse kappa light chain constant region frame (SEQ ID NO:32, SEQ ID NO:50) and cloned into pCDNA3.1+ carrier. The heavy chain plasmid and the light chain plasmid were then transiently transfected into CHO-S cells (Bibco, product number: A29133) in a 1:1 ratio. The resulting antibody was a recombinant mouse anti-human TIGIT antibody, named m10D8.

[0170] The m10D8 heavy chain V region sequence is shown in SEQ ID NO:20 and the light chain V region sequence is shown in SEQ ID NO:24. The m10D8 antibody HCDR1 sequence is SSYGMS (SEQ ID NO:21), the HCDR2 sequence is TINSNGGSTYYPDSVKG (SEQ ID NO:22), the HCDR3 sequence is LGTGTLGFAY (SEQ ID NO:23), the LCDR1 sequence is KASQDVKTAVS (SEQ ID NO:25), the LCDR2 sequence is WASTRHT (SEQ ID NO:26), and the LCDR3 sequence is QQHYSTPWT (SEQ ID NO:27).

[0171] The recombinantly expressed antibody was further verified by ELISA for its binding ability to human TIGIT (see FIG. 3), monkey TIGIT (see FIG. 4) and mouse TIGIT (see FIG. 5), with the positive control antibody being 10A7 antibody (hamster anti-mouse TIGIT, shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:28 and SEQ ID NO:30). The results reveal that m10D8 can bind well to human TIGIT and monkey TIGIT, but not to mouse TIGIT. The affinity of the m10D8 antibody was measured by Biacore-SPR instrument, revealing that the binding constant was 1.02E+06 (1 / Ms), the dissociation constant was 2.00E-04 (1 / s) and the equilibrium dissociation constant K D is 1.97E-10(M).

[0172] [Table 6]

[0173] JPEG0007628111000013.jpg237169

[0174] JPEG0007628111000014.jpg64169

[0175] Example 3 Ability of mouse anti-human TIGIT antibodies to block the binding of TIGIT to its ligand PVR Many studies have shown that blocking the binding of TIGIT to its ligand PVR by anti-TIGIT antibodies can restore the functions of effector T cells and NK cells, so it is necessary to study in vitro whether the binding of TIGIT to its ligand PVR can be blocked by selected mouse anti-human TIGIT.

[0176] An appropriate amount of PVR-Fc protein was taken from a -80°C refrigerator, and a biotin labeling reagent kit (EZ-Link® HSulfo-NHS-LC-Biotinylation Kit, product number: 21435) from Thermo Scientific was used to biotinylate PVR-Fc according to the operation steps in the instruction manual, and the labeled protein was named PVR-Fc-bio. The operation process of the experiment is as follows. 1) TIGIT-Fc (2 μg / ml) was coated, and the coating solution was 1×PBS (phosphate buffer solution), and the coating was performed overnight at 4°C. 2) Sealed with 3% BSA in a 37°C incubator for 2 h. 3) PVR-Fc-bio was diluted with PBS to a final concentration of 2 μg / ml, and this solution was used as the antibody diluent. 4) m10D8 and positive control antibody 10A7 antibody (hamster anti-mouse TIGIT, shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:28, SEQ ID NO:30), negative irrelevant control antibody were diluted with diluent to a starting concentration of 8 μg / ml, diluted half and half, 100 μl per well, incubated in a 37°C incubator for 2 h, and then washed 8 times with PBST (PBS containing 0.05% Tween 20). 5) Enzyme-labeled secondary antibody (Jackson Immuno Research Inc., product number: 016-030-084, diluted 1:10000) was added, 100 μl per well, incubated in a 37°C incubator for 1 h, and then washed 8 times with PBST. 6) Develop with TMB (3,3',5,5'-tetramethylbenzidine) developer, adding 100 μl per well. 7) 2M H 2 SO 4 The color development was stopped by adding 100 mM NaCl, and the values ​​were read at a wavelength of 450 nm using a microplate reader. The results are shown in Figure 6. Since the competitive inhibition curve of m10D8 is below that of the positive antibody 10A7 under the same conditions, it is clear that m10D8 has a stronger blocking ability against the binding of TIGIT to its ligand PVR than the positive antibody.

[0177] Example 4 Detection of biological functions of mouse anti-human TIGIT antibodies To simulate the complete activation of early T cells, both the first and second signals must be involved. Based on this principle, a method for detecting the biological activity of anti-TIGIT antibody was developed. The detection system includes two types of cells: an artificially constructed cell similar to an antigen-presenting cell (sAPC) that overexpresses mouse anti-human CD3 single chain antibody and PVR, and an engineered Jurkat cell (CTI-Jurkat) that overexpresses TIGIT and CD226 and is transfected with a luciferase reporter gene detection element whose transcription and translation are driven by an IL-2 promoter. The activation of T cells by the binding of anti-human CD3 single chain antibody on the sAPC cell membrane with the T cell receptor (TCR) on the Jurkat cell membrane belongs to the first signal, and the signal transduction by the interaction of PVR on the sAPC cell membrane with the activating receptor CD226 or the inhibitory receptor TIGIT on Jurkat cells belongs to the second signal. In the absence of anti-TIGIT antibodies, the affinity of PVR to TIGIT is much higher than that of PVR to CD226, so the preferential binding of PVR to TIGIT leads to inhibition of TIGIT intracellular structural domain signaling on T cell proliferation and cytokine release, and PVR blocks CD226 on the same cell surface from forming a functional dimer so as not to affect the normal physiological function of CD226. Furthermore, the affinity of CD226 to PVR is weak, so it cannot compete with TIGIT and PVR under the same conditions, resulting in ineffective activation of T cells, leading to T cells being in an overall inhibited state, and the IL-2 promoter cannot effectively initiate transcription and translation of the luciferase reporter gene.Conversely, when there is an anti-TIGIT antibody in the detection system, the high affinity anti-TIGIT antibody can bind to TIGIT to block the signal transduction between TIGIT and PVR, and TIGIT cannot block the dimerization of CD226, so that CD226 can effectively bind to PVR to transmit the intracellular structural domain signal of CD226 to activate T cells, and the IL-2 promoter can effectively initiate the transcription and translation of the luciferase reporter gene, and spontaneously emit a fluorescent signal to be captured by the instrument with the engagement of the luciferase substrate.

[0178] sAPC cells were prepared. CD CHO AGT TM sAPCs were cultured in medium (containing 25 μM MSX and 400 μg / ml hygromycin) until logarithmic growth phase, then centrifuged at 800 rpm for 5 min and resuspended in Ham's F-12 medium (containing 10% FBS) to 400,000 cells / ml, then plated into corresponding wells of a 96-well plate using a multichannel pipette, dispensing 100 μl per well, i.e., 40,000 cells per well, and incubated at 8% CO. 2 The cells were then placed in an incubator at 37° C. and cultured overnight. The next day, the medium was discarded and the cell culture plate was then placed face down on blotting paper to completely absorb the remaining medium.

[0179] CTI-Jurkat cells were prepared by culturing CTI-Jurkat cells in 1640 medium containing 10% FBS until log phase, then resuspending CTI-Jurkat cells to 5 million / ml in 1640 (1% FBS) medium, and adding the CTI-Jurkat cells to the sAPC wells with a multichannel pipette, 40 μl per well, i.e., 200,000 cells per well.

[0180] Antibodies were prepared. m10D8, positive control antibody Tiragolumab (shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:30) and negative control antibody BAT5906 (shown in Chinese patent application CN110003328A) were diluted with 1640 (1% FBS) medium to three concentrations of 64, 32, 16 μg / ml, and 0 μg / ml as a control. The diluted antibodies were then added to the wells containing sAPCs and CTI-Jurkat cells, with 40 μl per well (i.e., the final antibody concentrations were 32, 16, 8, 0 μg / ml).

[0181] The cell culture plate containing the sample was incubated at 8% CO 2 The plate was incubated in a 37°C incubator for 4 hours, then removed and placed at room temperature for 10 minutes to allow the cell culture plate to recover to room temperature. Then, luciferase substrate (taken out of the -20°C refrigerator 2 hours before and placed at room temperature) was added to the corresponding sample wells, 80 μl per well. The culture plate was placed in a microplate reader, shaken for 5 s, and then read out the fluorescence value after 10 minutes, as shown in Figure 7. The results show that both Tiragolumab and m10D8 have the function of activating T cells, but under the same conditions, m10D8 has the strongest ability to activate T cell function.

[0182] Example 5 Humanization of mouse anti-human TIGIT antibody The heavy and light chain variable regions of the mouse anti-human TIGIT antibody were compared with the sequences in the human antibody database, and the results are shown in Table 5.1. The humanization process must ensure high affinity of the antibody while also ensuring drug discovery potential and potential immunogenicity of the drug. In conjunction with bioinformatics modeling calculations, the first humanization of m10D8 humanized antibody was designed with three mutants, namely h10D8V1 (shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:29, SEQ ID NO:30), h10D8V2 (shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:28, SEQ ID NO:30), and h10D8V3 (shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:28, SEQ ID NO:30). The light chain variable region frame of h10D8V1 and h10D8V2 is IGKV4-1*01, and the heavy chain variable region frame is IGHV3-23*01. The heavy chain variable region frame of h10D8V3 is IGHV3-64*04, and the light chain variable region frame is IGKV1-NL1*01. Binding experiments were performed with the mutton protein and TIGIT, and biological activity was detected. Binding experiments were performed with purified h10D8V1, h10D8V2, and h10D8V3 and TIGIT. As shown by the ELISA results (shown in Figure 8), the EC50 (concentration that induces 50% of the maximum effect) of the three antibodies was 0.07623 μg / ml, 0.09241 μg / ml, and 0.05259 μg / ml, respectively. Judging from the EC50, h10D8V3 appears to have a stronger binding ability with TIGIT. However, the static binding status of the antigen and antibody reflected in the ELISA results cannot dynamically reflect the binding and dissociation status of the antibody and antigen. Therefore, the equilibrium dissociation constant K Dwas measured (shown in FIG. 9 and Table 5.2). The results show that h10D8V3 has a slow binding rate compared to the other two antibodies, but a fast dissociation rate and a larger equilibrium dissociation constant than the other two antibodies, i.e., its affinity is smaller than the other two antibodies. The variable regions of h10D8V1 and h10D8V2 are completely identical, and differ only by two amino acid residues at positions 356 (EU numbering, corresponding Kabat number: position 377) and 358 (EU numbering, corresponding Kabat number: position 381) in the constant region, so there is no significant difference in affinity. At the same time, the biological activation methods of h10D8V1, h10D8V2 and h10D8V3 were further compared (see Example 4 for the method), and the results (shown in Figure 10) revealed that the activation abilities of h10D8V1, h10D8V2 and h10D8V3 on T cells are relatively similar, indicating that the biological activities of the three are similar.

[0183] [Table 7]

[0184] [Table 8]

[0185] Based on the results of h10D8V1, h10D8V2, and h10D8V3, a second round of humanization design was further carried out on m10D8, resulting in three further mutons, namely h10D8OF (shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:28, SEQ ID NO:30, i.e., the heavy chain sequence is SEQ ID NO:44, and the light chain sequence is SEQ ID NO:45), h10D8V4 (shown in SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:28, SEQ ID NO:30), and h10D8V5 (shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:28, SEQ ID NO:30). The heavy chain frame of h10D8OF is IGHV3-64*04, and the frame used for the light chain is IGKV3D-7*01. The heavy chain frame of h10D8V4 is IGHV3-64*04, and the light chain frame is IGKV4-1*01, with the main feature being that some amino acid residues in the frame match those of the mouse antibody m10D8. The heavy and light chain frames of h10D8V5 are similar to h10D8V4, and the main feature is that the antibody-antigen interface matches as closely as possible with the mouse antibody m10D8 and TIGIT interfaces. The binding abilities of h10D8OF, h10D8V4, and h10D8V5 with TIGIT were compared side by side, and the results, as shown in Figure 11, reveal that the binding curves of h10D8OF, h10D8V4, and h10D8V5 are essentially overlapping, with EC50 values ​​of 0.02329 μg / ml, 0.02560 μg / ml, and 0.02408 μg / ml, respectively.

[0186] At the same time, the binding ability of h10D8OF and h10D8V2 with TIGIT was compared. The degree of polymerization of the binding curves of both was high, and the EC50 value of h10D8OF was slightly smaller than that of h10D8V2. Based on the ELISA results alone, it was shown that the affinity of the former may be slightly higher (shown in FIG. 12). The EC50 values ​​of h10D8OF, h10D8V2 and Tiragolumab were 0.04273 μg / ml, 0.05310 μg / ml and 0.05361 μg / ml, respectively. In addition, the equilibrium dissociation constant K of h10D8OF, h10D8V2, Tiragolumab and TIGIT-his tag antigen was determined by SPR-Biacore technology. D (shown in Figure 13 and Table 5.3), dynamic affinity detection can find that the affinity of h10D8V2 with monovalent TIGIT-his is higher than that of h10D8OF. At the same time, the biological activity of h10D8OF, h10D8V4, and h10D8V5 was further detected, the detection method was similar to that in Example 4, and the results (shown in Figure 14) reveal that at high concentration (32μg / ml), h10D8OF has a stronger ability to restore T cell function, but the results of h10D8V4 and h10D8V5 are very close.

[0187] [Table 9]

[0188] [Table 10]

[0189] JPEG0007628111000019.jpg247169

[0190] Example 6 Blocking experiment of the binding of TIGIT to PVR by humanized antibodies After mouse antibody is humanized, theoretically, some functions of mouse antibody such as blocking the binding of TIGIT and PVR are still retained. The method of the experiment of blocking the binding of TIGIT and PVR by humanized antibody is similar to that in Example 3. By ELISA method, the blocking ability of h10D8OF, h10D8V4, h10D8V5, Tiragolumab (positive control antibody), and irrelevant antibody (isotype control) BAT5906 to the binding of TIGIT and PVR under the same conditions is detected. The experimental results (shown in Figure 15) show that h10D8OF, h10D8V4, h10D8V5 and Tiragolumab can effectively block the binding of TIGIT and PVR, with IC50 values ​​(concentration at which 50% inhibition occurs) of 0.1592μg / ml, 0.2166μg / ml, 0.2837μg / ml and 0.4289μg / ml, respectively. From the IC50 values ​​and the upper and lower platform values ​​of the four-parameter curve, the blocking ability of h10D8OF and h10D8V4 to the binding of TIGIT and PVR is stronger than that of the positive antibody Tiragolumab.

[0191] In addition, the blocking ability of these antibodies to the binding of biotinylated PVR to TIGIT on the surface of Jurkat cells was verified again by using Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT 2E6). The antibody was diluted with PBS to prepare a biotinylated PVR-Fc protein solution with a concentration of 80 nM as the antibody dilution solution. The starting concentration of the antibody was 15 nM, and it was diluted half and half to provide a total of 10 concentration gradients (including 0 nM). The 10 concentration gradients of the antibody were divided and packed into 10 centrifuge tubes each containing 1 million Jurkat-hTIGIT cells, and the cells were resuspended and incubated at 4°C for 1 hour. The cells in each tube were then centrifuged to remove the supernatant and washed twice with PBS. Then, 100μ of Streptavidin PE (Thermo, product number: 12-4317-87) diluted 1:2000 was added to each tube, resuspended, and incubated at 4℃ for 40 minutes. Then, the cells in each tube were centrifuged to remove the supernatant, washed twice with PBS, and resuspended in 200μl of PBS, and the fluorescence value (mean FL2-A) of the cells was detected by a flow analyzer. Finally, the collected fluorescence value was analyzed by a four-parameter fitting graph to calculate the IC50 value. The IC50 values ​​of h10D8OF, h10D8V4, and h10D8V5 (shown in FIG. 16) are 11.26nM, 15.78nM, and 10.44nM, respectively, and all three antibodies can effectively block the binding of biotinylated PVR-FC and TIGIT on the membrane surface of Jurkat-hTIGIT cells, but the blocking effect of h10D8OF and h10D8V5 is better. At the same time, the blocking ability of h10D8OF and the positive antibody Tiragolumab to the binding of PVR and TIGIT was compared (shown in FIG. 17), and the blocking effect of h10D8OF was stronger.

[0192] Example 7 Detection of humanized antibody ADCC activity Jurkat cells (ATCC, Clone E6-1, product number: TIB-152 TMThe stable cell line was constructed by transfecting NFAT and luciferase reporter gene elements and FcγRIIIa (CD16) receptor gene into the monoclonal antibody, which stably expresses FcγRIIIa receptor, a necessary molecule for mediating the biological activity of ADCC. The monoclonal antibody connects the effector cells and the target cells, activates the NFAT pathway, and initiates the expression of the luciferase reporter gene. The stable cell line was constructed by transfecting Jurkat cells (ATCC, Clone E6-1) with the human TIGIT gene so that the human TIGIT molecule was overexpressed, which was the target cell. The process of detecting the antibody ADCC activity is as follows: 1) According to the cell density, an appropriate amount of cells was taken, and the growth medium was removed by centrifugation at 1200 rpm for 5 minutes, and the cell density was adjusted. 2) The two types of cells were evenly spread on a cell culture plate (Corning, Product No.: 3917). The effector cells were 80,000 cells / well, and the target cells were 20,000 cells / well, with a total volume of 50 μl for the two types of cells per well. 3) The antibodies were diluted using 1% FBS1640 as a dilution medium. h10D8OF, Tiragolumab, and an irrelevant antibody (without ADCC activity) were diluted 1 / 3 with a starting concentration of 1.8 μg / ml (50 μl per well, final concentration of 0.9 μg / ml), with three duplicate wells for each concentration gradient, and the final concentration gradient was 0 μg / ml. 4) After adding the antibodies, the plate was tapped lightly to ensure the samples were mixed evenly. Then, the plates were incubated at 37°C in 8% CO 2 Place in an incubator and incubate for 4 hours. 5) After incubation, leave at room temperature for 10 minutes, and allow luciferase substrate reagent to equilibrate to room temperature. Add 100μl of substrate reaction solution to each well, and read the value after 10 minutes. The results are shown in Figure 18. Both h10D8OF and Tiragolumab have ADCC activity, and under the same conditions, the ADCC activity of h10D8OF is slightly better than that of Tiragolumab.

[0193] In another experiment, the host cell CHO secreting h10D8OF was modified (the modification method is shown in patent CN107881160A and WO2019029713 A1), and the fucose of the h10D8OF antibody was removed, and the antibody was named h10D8OFKF. By a method similar to the above, the difference in ADCC activity between h10D8OFKF and h10D8OF was compared. The results are shown in Figure 19, and both h10D8OFKF and h10D8OF have ADCC activity, with EC50 of 0.0152nM and 0.125nM, respectively. Under the same conditions, the ADCC activity of h10D8OFKF is 8.8 times that of h10D8OF, and stronger ADCC activity may have better efficacy in the body.

[0194] Example 8 Cytokine release experiment under the action of humanized anti-TIGIT antibody After TIGIT binds to PVR (CD155) expressed on the surface of tumor cells or APC cells, the effector function of T cells is reduced, inhibiting antitumor immune responses. The blocking ability of anti-TIGIT antibodies to inhibit PVR-mediated T cell cytokine release was detected. Purchased human primary CD3+ cells (Guangzhou Leide Biotechnology Co., Ltd., product number: 1507) were induced in 10% FBS1640 medium containing PHA (2 μg / ml) and IL-2 (4 ng / ml) and cultured for 10 days, then the cells were centrifuged and resuspended in medium without PHA and IL-2, and then incubated at 4 °C for 10 min at 5% CO 2 The cells were then placed in a 37°C cell incubator and cultured stationarily for 24 hours. Then, the cells were diluted with 5 × 10 5 After resuspending at 1000μg / ml, the cells were incubated with irrelevant antibody BAT5906 (as shown in Chinese patent application CN110003328A), h10D8OF (25μg / ml and 10μg / ml), and tiragolumab (25μg / ml and 10μg / ml) at room temperature for 30 min. After the incubation was completed, the cells were seeded onto cell culture plates pre-coated with anti-CD3 antibody (10μg / ml) (BD Biosciences, product number 555336), PVR-Fc (10μg / ml), or anti-CD3 antibody (10μg / ml) and PVR-Fc (10μg / ml), and then placed in a cell incubator (37°C, 5% CO).2 ) and cultured for 48 hours. Finally, the cells in the cell culture plate were removed and centrifuged to retain the supernatant. After diluting the supernatant to a certain number of times, the release of cytokines IFN-γ and IL-2 in the cell supernatant was detected.

[0195] The cytokine production induced by lymphocytes with anti-CD3 antibody alone was compared to the cytokine production induced by other treatments, the former set as 1.0 and the latter shown as actual ratios, and the results are shown in Figures 20 and 21. The results show that h10D8OF can induce lymphocytes to produce more IFN-γ and IL-2 with increasing concentration.

[0196] Example 9: Verification of the tumor-inhibiting ability of anti-TIGIT antibodies using a model in which B-hPD-1 / hTIGIT double-humanized mice were inoculated with MC38 tumors Since humanized antibodies do not recognize mouse TIGIT, the extracellular domains of PD-1 and TIGIT molecules in the mouse genome on a C57BL / 6 background were humanized to replace them with human PD-1 and human TIGIT, i.e., B-hPD-1 / hTIGIT double humanized mice, the strain of which was C57BL / 6-Pdcd1 tm1(hPDCD1)Tigit tm1(hTIGIT) / Bcgen (Bcgen Genetics Biotechnology Co., Ltd.). In splenocytes of B-hPD-1 / hTIGIT homozygous mice, the expression of hTIGIT and hPD-1 mRNA could be detected, but the expression of mTIGIT and mPD-1 mRNA could not be detected (data from the official website of Bcgen Genetics Biotechnology Co., Ltd.). By creating an animal model of MC38 colon cancer using B-hPD-1 / hTIGIT humanized mice, we examined the efficacy of anti-TIGIT antibodies and the combined efficacy of anti-TIGIT and anti-PD-1 antibodies.

[0197] Prior to the animal pharmacological experiment, a preliminary experiment was conducted to verify that the anti-TIGIT antibody could effectively bind to hTIGIT on the surface of cells in the body of B-hPD-1 / hTIGIT double humanized mice. 11.5 μg of anti-mCD3e antibody (Biolegend, product number: 100301) was dissolved in 300 μl of PBS, and then one B-hPD-1 / hTIGIT double humanized mouse was picked and intraperitoneally injected with 200 μl of anti-mCD3e antibody solution. After 24 h, the mouse was killed by cervical dislocation, and the mouse spleen was rapidly removed and placed in a 15 ml centrifuge tube to which 5 ml of PBS had been added. The spleen was then transferred to a 6-well plate, placed on a 70 μm sieve, 1 ml of PBS was added, polished with the tail of a disinfected syringe, and the sieve was washed with 1 to 2 ml of PBS. The filtered cell suspension was centrifuged at 3000 rpm for 5 min, the supernatant was removed, and then centrifuged briefly to remove the supernatant. Then, 1 ml of red blood cell lysis solution (Biolegend, product number: C3702) was added to each spleen and lysed on ice for 5 min. Finally, 5 ml of PBS was added, the cells were mixed uniformly, and centrifuged at 3000 rpm for 3 min at 4 °C, the supernatant was removed, and then centrifuged briefly to remove the supernatant. Then, detection was performed with a flow analyzer. First, the cells were mixed and suspended in 300 μl of PBS, and 6 μl of anti-mCD16 / 32 antibody (Biolegend, #101310) and Human TruStrain FcX (Biolegend, product number: 422302) were added and mixed uniformly, and incubated on ice for 15 min. 1.2 ml of PBS was added to mix the cells uniformly, and the cell concentration was adjusted to 4 × 10 7The cell suspension was adjusted to 1 / ml, and 25 μl of the cell suspension was added to the tube to be stained. Each tube was treated with the corresponding antibodies PerCP-anti-mTcRβ (Biolegend, product number: 109228) and APC-anti-hTIGIT antibody APC-anti-hTIGIT (eBioscience, product number: 17-9500-42), mouse anti-TIGIT antibody secondary antibody Anti-mouse IgG with Alexa Fluor 647 (Beijing Boaoxin Biotechnology Co., Ltd., product number: bs-0296G-AF647), Goat anti-Human IgG Fc Secondary Antibody with FITC, and humanized anti-TIGIT antibody secondary antibody anti-human Fc with FITC (Invitrogen, product number A18818), and then placed on the instrument for detection. The experimental design is shown in Table 9.1, and the flow analysis results (shown in Figure 22) show that m10D8, h10D8V1, h10D8OF, and Tiragolumab can effectively bind to hTIGIT on the surface of B-hPD-1 / hTIGIT double humanized mouse T cells, among which h10D8OF has stronger binding ability to hTIGIT than other antibodies under the same conditions. A similar situation exists in the binding experiment between h10D8OF, Tiragolumab, and Jurkat-hTIGIT 2E6, a Jurkat cell overexpressing human TIGIT (shown in Figure 23), and it can be shown that h10D8OF (EC50 value is 0.6961 nM) and TIGIT in physiological state or TIGIT close to physiological state have stronger affinity compared to Tiragolumab (EC50 value is 1.906 nM).

[0198] The mice were 6-8 weeks old and all female mice were selected, with n = 6 mice per group. MC38 cells (Baiao Saitu Gene Biotechnology Co., Ltd.) were inoculated subcutaneously into the right axilla of the mice at 5 × 10 5 The tumor volume was 150±50 mm 3At this stage, the mice were divided into groups and administered, and tumor-bearing mice with tumors that were too large or too small were eliminated. The mice were administered once every 3 days, and the tumor volumes were measured twice a week. The dose of Pembrolizumab was 0.1 mg / kg, and the doses of Tiragolumab and m10D8 were both 20 mg / kg each time the mice were administered. The efficacy results of the single-agent anti-TIGIT antibody (shown in FIG. 24) show that both Tiragolumab and m10D8 antibodies can inhibit tumor growth, and the efficacy of the single-agent m10D8 and Tiragolumab is relatively similar. The efficacy results of the single-agent humanized anti-TIGIT antibody (shown in FIG. 25) show that the tumor inhibitory effect of h10D8V2 is low, and the tumor inhibitory effect of h10D8OF and Tiragolumab is relatively similar. The efficacy results of anti-TIGIT antibody and Pembrolizumab combination (shown in Figure 26) show that the tumor inhibition effect of h10D8V2 and Pembrolizumab combination is poor, and the efficacy of h10D8OF and Pembrolizumab combination is relatively close to that of Tiragolumab and Pembrolizumab combination. In summary, from the viewpoint of efficacy in animals, both mouse antibody and humanized antibody have the effect of inhibiting tumor growth, and have potential effects when combined with Pembrolizumab.

[0199] [Table 11]

[0200] Example 10: Effects of humanized anti-TIGIT antibodies on lymphocyte subpopulations When performing the therapeutic evaluation item of the in vivo pharmacological efficacy test, the effect of anti-TIGIT antibody on CD4+T cells and CD8+T cells in tumor infiltrating lymphocytes (TIL) of mouse MC38 was analyzed. For the test, tumors of the vehicle control group (injectable saline) (n=4) and the h10D8OF (20mg / kg) single agent treatment group (n=5) were selected, and fresh tumor tissues of the mice were surgically excised, washed with PBS, and stored on ice. After that, they were repeatedly sheared with ophthalmic scissors, placed in an IKA polisher, rotated forward and backward at 1200 rpm for 10 s, and polished for a total of 2 min. The polished tumor tissue solution was filtered into a 50 mL centrifuge tube with a 70 μm sieve, and the tissue was washed once, and then centrifuged at 4 ° C, 2000 rpm for 5 min in a low-temperature centrifuge, the supernatant was removed, and the tumor cells were resuspended in an appropriate amount of PBS.

[0201] First, dead / live cells were stained with 2 μl of Zombie NIR. TMA dye (Biolegend #423106) was taken and added to 500μl of PBS to prepare 2× dead / live cell staining solution. A 96-well plate was taken, and 25μl of tumor cell suspension was added to each well of the experimental group, and 25μl of 2× dead / live cell staining solution was taken and added to the corresponding well containing the tumor cell suspension, and then incubated for 15min with gentle shaking in a dark room temperature environment. Next, the cell surface was stained, and 25μl of the cells stained in the previous step were transferred to a new 96-well plate with a pipette, and 25μl of cell surface labeling molecule staining solution was added per well, and incubated at 4℃ in the dark for 30min. The staining solution for cell surface marker molecules included anti-CD45 antibody (FITC, 0.5 μl, Biolegend #231092), anti-mCD4 antibody (PE, 0.5 μl, Biolegend #248731), anti-mCD8 antibody (BV510, 0.5 μl, Biolegend #248151), and anti-hTIGIT antibody (APC, 1 μl, Biolegend #4272773). After the end of the incubation, 185 μl of PBS was added to resuspend the cells, mixed uniformly, and centrifuged at 2000 rpm at 4°C for 5 min, the supernatant was removed, and the plate was gently inverted on blotting paper to further remove the residual liquid. Then, 200 μl of fixative (1×) was added per well to resuspend the cells, mixed uniformly, and left at 4°C overnight. After the fixation, the plate was centrifuged at 3000 rpm for 5 min, the supernatant was removed, and the plate was gently inverted on blotting paper to remove the remaining liquid. 200 μl of membrane penetration solution was added per well, centrifuged at 3000 rpm for 5 min, the supernatant was removed, and the plate was gently inverted on blotting paper to remove the remaining liquid. 25 μl of membrane penetration solution was added per well to resuspend the cells, and 25 μl of the prepared intracellular staining solution (Anti-Mouse / Rat Foxp3, APC) was added, and the plate was incubated at 4 ° C in the dark for 30 min. 200 μl of 1 × membrane penetration solution was added per well, mixed uniformly, and centrifuged at 4 ° C, 3000 rpm for 5 min, the supernatant was removed, and the plate was gently inverted on blotting paper to remove the remaining liquid. 250 μl of PBS was added to resuspend the cells, and the plate was placed on the instrument and detected according to the SOP of the Attune NXT flow cytometry.

[0202] First, mCD45+ leukocytes (shown in FIG. 27) were selected from the prepared single cells, and CD4+T (shown in FIG. 28) and CD8+T (shown in FIG. 29) cells in the mCD45+ cells were analyzed. The results showed that there was no significant difference in mCD45+ lymphocytes between the two groups of mouse TILs, and further analysis revealed that there was no significant difference in CD4+T and CD8+T cells. Next, TIGIT+CD4+T cells and TIGIT+CD8+T cells were further analyzed (shown in FIG. 30 and 31), and it was found that the proportion of TIGIT+CD4+T and TIGIT+CD8+T cells was significantly reduced in the single agent treatment group compared to the control group. The results suggest that under the action of anti-TIGIT antibody, the expression of TIGIT molecule is likely to be reduced in the CD4+T and CD8+T cells of mouse colon cancer MC38 tumor TILs, and indirectly suggest that the function of CD8+T cells was restored.

[0203] Example 11: Verification of the tumor-inhibiting ability of anti-TIGIT humanized antibodies using B-hTIGIT humanized mice and hPD-L1 MC38 tumor models Because anti-TIGIT humanized antibodies do not recognize mouse TIGIT, the extracellular domain of the TIGIT molecule in the mouse genome on the C57BL / 6 background was humanized and replaced with human TIGIT, i.e., B-hTIGIT humanized mice. The mouse strain was C57BL / 6-Tigit tm1(hTIGIT) / Bcgen (Bcgen Genetics Co., Ltd.). In splenocytes from B-hTIGIT homozygous mice, hTIGIT mRNA expression was detectable, but mTIGIT mRNA expression was not detectable (data from the official website of Bcgen Genetics Co., Ltd.). This experiment used B-hTIGIT humanized mice and an MC38 tumor model overexpressing hPD-L1 to verify the tumor-inhibiting ability of anti-TIGIT humanized antibodies, as well as the potential synergistic effect of combined administration of anti-TIGIT and anti-PD-L1 antibodies.

[0204] In order for h10D8OF and Tiragolumab to most effectively perform ADCC function in mice, the heavy chain constant regions of h10D8OF and Tiragolumab were replaced with the heavy chain constant region of mouse antibody IgG2a (SEQ ID NO:31), and the light chain constant regions of h10D8OF and Tiragolumab were replaced with the light chain constant region of mouse antibody IgG2a (SEQ ID NO:32), and the recombined antibodies were named mh10D8OF and mTiragolumab.

[0205] Six to eight-week-old B-hTIGIT humanized mice were selected and 5 × 10 MC38-hPD-L1 cells (MC38 cells expressing human PD-L1 and depleting mouse PD-L1) (Baiaosaitu Gene Biotechnology Co., Ltd.) resuspended in PBS were added to the mice. 5 The tumor was subcutaneously inoculated into the right side of B-hTIGIT humanized mice at a concentration of 0.1 mL / cell and a volume of 0.1 mL / mouse. The mean tumor volume was 100-150 mm. 3 When the tumor volume and body weight reached 10, mice with appropriate tumor volume and body weight were grouped and equally assigned to six experimental groups with 10 mice per group. The administration route was intraperitoneal injection. The control group was Vehicle (saline for injection), and there were two groups of mh10D8OF, with doses of 10mg / kg and 30mg / kg, respectively. The dose of mTiragolumab was 30mg / kg, and the dose of the anti-PD-L1 antibody Atezolizumab was 1mg / kg. The doses of the combination administration group were mh10D8OF 30mg / kg and anti-PD-L1 antibody Atezolizumab 1mg / kg. The experimental results are shown in Figure 32.

[0206] Both mh10D8OF and mTiragolumab can effectively inhibit tumor growth, and there is no statistical difference in the degree of inhibition, with both TGIs being above 50%. TGI (The Tumor Growth Inhibition value) is the tumor (volume) inhibition rate. It is used to evaluate the inhibitory effect of test drugs on tumor growth in vivo (i.e., animal experiments). It can be calculated using the formula (100-T / C×100)%, where T is the average relative tumor volume after treatment and C is the average relative tumor volume before treatment. Due to the drug dose setting, no significant synergistic effect was observed in this study, but biologically, the degree of inhibition of tumors by combined administration under the conditions of the dose was more significant, with the tumor inhibition rate TGI being 66%. [Brief description of the drawings]

[0207] [Figure 1] FIG. 1 is a schematic diagram showing the construction of a PVR-FC fusion protein. [Diagram 2] FIG. 1 is a schematic diagram of the construction of a TIGIT-FC fusion protein. [Diagram 3] These are the results of an ELISA experiment between m10D8 antibody and human TIGIT. [Figure 4] These are the results of an ELISA experiment between m10D8 antibody and monkey TIGIT. [Diagram 5] These are the results of an ELISA experiment between m10D8 antibody and mouse TIGIT. [Figure 6] These are the results of an experiment to block the binding of TIGIT to PVR by mouse anti-human TIGIT. [Figure 7] This shows the results of detecting the biological activity of mouse anti-human TIGIT antibody. [Figure 8] This is the result of comparing the binding ability of h10D8V1, h10D8V2, and h10D8V3 to TIGIT. [Figure 9] The results show the results of measuring the equilibrium dissociation constants of h10D8V1, h10D8V2, and h10D8V3 with TIGIT-his tag. [Figure 10]This is the result of comparing the biological activities of h10D8V1, h10D8V2, and h10D8V3. [Figure 11] This is the result of comparing the binding ability of h10D8OF, h10D8V4, and h10D8V5 to TIGIT. [Figure 12] This is the result of comparing the binding ability of h10D8OF and h10D8V2 to TIGIT. [Figure 13] This shows the results of detecting the equilibrium dissociation constants of h10D8OF, h10D8V2, and the TIGIT-his tag antigen. [Figure 14] This is the result of comparing the biological activities of h10D8OF, h10D8V4, and h10D8V5. [Figure 15] These are the results of an experiment blocking the binding of TIGIT to PVR by h10D8OF, h10D8V4, and h10D8V5. [Figure 16] This shows the results of comparing the ability of h10D8OF, h10D8V4, and h10D8V5 to block the binding of PVR to TIGIT on the membrane surface of Jurkat cells. [Figure 17] The results show a comparison of the ability of h10D8OF and tiragolumab to block the binding of PVR to TIGIT on the membrane surface of Jurkat cells. [Figure 18] This shows the results of detecting h10D8OF ADCC activity. [Figure 19] This shows the results of comparing the ADCC activities of h10D8OF and h10D8OFKF. [Figure 20] Relative fold induction of IFN-γ, 10 indicates 10 μg / ml and 25 indicates 25 μg / ml. [Figure 21] Relative fold induction of IL-2, 10 indicates 10 μg / ml and 25 indicates 25 μg / ml. [Figure 22] These are the results of a binding experiment between anti-TIGIT antibodies and hTIGIT on the surface of B-hPD-1 / hTIGIT double humanized mouse T cells. The secondary antibodies for the humanized antibodies are anti-human Fc and FITC, and the secondary antibodies for the mouse antibodies are anti-mouse IgG and Alexa Fluor 647. [Diagram 23] This shows the results of comparing the binding abilities of h10D8OF and tiragolumab to TIGIT on the surface of Jurkat-hTIGIT cells. [Figure 24] This is the efficacy of mouse antibody alone. [Diagram 25] This is the efficacy of co-administering humanized antibodies. [Figure 26] This is the efficacy of co-administering humanized antibodies. [Figure 27] Percentage of live mCD45 cells in MC38 tumor-infiltrating lymphocytes. [Figure 28] Percentage of CD4+ T cells among live mCD45 cells. [Figure 29] Percentage of CD8+ T cells among live mCD45 cells. [Diagram 30] This is the percentage of TIGIT+CD4+ T cells among live CD4+ T cells. [Diagram 31] This is the percentage of TIGIT+CD8+ T cells among live CD8+ T cells. [Diagram 32] These are the results of in vivo pharmacological experiments.

Claims

1. The antibody specifically binds to the T cell immunoreceptor having an immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (TIGIT) protein, and has a VH CDR1 shown in SEQ ID NO:21, a VH CDR2 shown in SEQ ID NO:22, a VH CDR3 shown in SEQ ID NO:23, a VL CDR1 shown in SEQ ID NO:25, a VL CDR2 shown in SEQ ID NO:26, a VL CDR3 shown in SEQ ID NO:27, a VL CDR4 shown in SEQ ID NO:29, a VL CDR5 shown in SEQ ID NO:30, a VL CDR6 shown in SEQ ID NO:31, a VL CDR7 shown in SEQ ID NO:32, a VL CDR8 shown in SEQ ID NO:33, a VL CDR9 shown in SEQ ID NO:34, a VL CDR10 shown in SEQ ID NO:35, a VL CDR An antibody or antigen-binding fragment thereof, comprising a VL CDR2 as shown in SEQ ID NO: 26 or 43 and a VL CDR3 as shown in SEQ ID NO:

27.

2. VH CDR1 shown in SEQ ID NO: 21, VH CDR2 shown in SEQ ID NO: 22, VH CDR3 shown in SEQ ID NO: 23, VL CDR1 shown in SEQ ID NO: 25, VL CDR1 shown in SEQ ID NO: 43 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a VL CDR2 and a VL CDR3 as set forth in SEQ ID NO:

27.

3. 2. The antibody or antigen-binding fragment thereof of claim 1, which is of one of the following isotypes: IgG, IgM, IgA, IgE or IgD.

4. The antibody or antigen-binding fragment thereof of claim 1, further comprising a heavy chain constant region, a light chain constant region, or an Fc region.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the Fc region is a human Fc region.

6. The antibody or antigen-binding fragment thereof of claim 5, characterized in that the human Fc region has an E amino acid at position 356 according to the EU numbering system.

7. The antibody or antigen-binding fragment thereof of claim 5, characterized in that the human Fc region has an amino acid of M at position 386 according to the EU numbering system.

8. The antibody or antigen-binding fragment thereof of claim 5, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or 29.

9. The antibody or antigen-binding fragment thereof of claim 5, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain, the light chain comprising an amino acid sequence as set forth in SEQ ID NO:

30.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is a humanized antibody.

11. The antibody or antigen-binding fragment thereof of claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 33, 35, 37, 39 and / or 41, or a peptide having at least 90% sequence identity to an amino acid sequence in the group consisting of SEQ ID NO: 20, 33, 35, 37, 39 and / or 41.

12. The antibody or antigen-binding fragment thereof of claim 1, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 34, 36, 38, 40 and / or 42, or a peptide having at least 90% homology to an amino acid sequence in the group consisting of SEQ ID NO: 24, 34, 36, 38, 40 and / or 42.

13. An antibody or antigen-binding fragment thereof that specifically binds to immunoglobulin and T cell immunoreceptor having immunoreceptor tyrosine-based inhibitory motif (TIGIT) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:35 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:

36.

14. The antibody or antigen-binding fragment thereof according to any one of claims 11 to 13, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 28 and the light chain comprises the amino acid sequence shown in SEQ ID NO:

30.

15. The antibody or antigen-binding fragment thereof according to any one of claims 11 to 13, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 29 and the light chain comprises the amino acid sequence shown in SEQ ID NO:

30.

16. An antibody or antigen-binding fragment thereof that specifically binds to immunoglobulin and T cell immunoreceptor having immunoreceptor tyrosine-based inhibitory motif (TIGIT) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:44 and a light chain having the amino acid sequence set forth in SEQ ID NO:

45.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, which is capable of effectively blocking the binding between TIGIT and PVR.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, which is capable of activating lymphocytes to release cytokines.

19. Affinity value K for human TIGIT Ｄ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof has a binding affinity of 100 pM or less.

20. Affinity value K for human TIGIT Ｄ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof has a binding activity of 5 nM or less.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, which has ADCC activity.

22. 22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, which is not bound to fucose.

23. A bispecific antibody, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22 and a second antigen-binding fragment having specificity for a molecule in an immune cell.

24. 24. The bispecific antibody of claim 23, wherein the molecule comprises PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR, or CD47.

25. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a bispecific antibody according to claim 23 or 24, and a pharma- ceutically acceptable carrier.

26. A cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the bispecific antibody of claim 23 or 24.

27. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a bispecific antibody according to claim 23 or 24, wherein the pharmaceutical composition is used for the treatment of cancer or an infectious disease.

28. The pharmaceutical composition of claim 27, wherein the cancer is a solid tumor.

29. The pharmaceutical composition of claim 27, characterized in that the cancer is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer and thyroid cancer.

30. The pharmaceutical composition described in any one of claims 27 to 29, further comprising a second cancer therapeutic agent.

31. 28. The pharmaceutical composition of claim 27, wherein the infection is a viral infection, a bacterial infection, a fungal infection or a parasitic infection.

32. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the bispecific antibody of claim 23 or 24, said pharmaceutical composition being used for the treatment of cancer or an infectious disease, said treatment comprising: (a) treating cells in vitro with the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the bispecific antibody of claim 23 or 24; (b) administering the treated cells to a patient; and A pharmaceutical composition comprising:

33. The pharmaceutical composition of claim 32, wherein the treatment further comprises isolating the cells from an individual prior to step (a).

34. 34. The pharmaceutical composition of claim 33, wherein the cells are isolated from within the patient's body.

35. 34. The pharmaceutical composition of claim 33, wherein the cells are isolated from a donor individual different from the patient.

36. The pharmaceutical composition according to any one of claims 32 to 35, characterized in that the cells are T cells.

37. The pharmaceutical composition of claim 36, wherein the T cells are tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

38. A method for detecting TIGIT expression in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a bispecific antibody described in claim 23 or 24, thereby allowing the antibody or antigen-binding fragment thereof to bind to TIGIT, and detecting the binding, which reflects the expression level of TIGIT in the sample.

39. 40. The method of claim 38, wherein the sample comprises tumor cells, tumor tissue, infected tissue or a blood sample.

40. A polynucleotide capable of encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 22, or a bispecific antibody according to claim 23 or 24.

41. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a bispecific antibody according to claim 23 or 24, characterized in that a cell comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof is cultured to express the antibody or antigen-binding fragment thereof.

42. 42. The method of claim 41, wherein the cell is a CHO cell, a HEK293 cell, a Cos1 cell, a Cos7 cell, a CV1 cell or a mouse L cell.

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