HK40068895B - Anti-tigit immunosuppressant and application thereof - Google Patents

Description

An anti-TIGIT immunosuppressant and its application

Technical Field

This invention relates to the technical field of immunology. Specifically, it relates to immune checkpoint antibodies, their preparation, and their immunological applications.

Background Technology

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a T cell immune receptor with immunoglobulin (Ig) and tyrosine inhibitor motif (ITIM) domains, primarily 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, also known as VSIG9, VSTM3, and WUCAM, has a structure that shows it contains an extracellular immunoglobulin domain, a type I transmembrane region, and two ITIM motifs. TIGIT is part of a co-stimulatory network, primarily composed of the activating receptor CD226 and the inhibitory receptor TIGIT on T cells, as well as ligands CD155 (also known as PVR, a poliovirus receptor protein encoded by the PVR gene in humans) and CD112 expressed on the surface of APCs, tumor cells, and infected cells. Binding of TIGIT to PVR or CD112 induces phosphorylation of Tyr225 in the TIGIT cytoplasm, leading to TIGIT binding to cell adaptive growth factor receptor-binding protein 2 (GRB2). GRB2 recruits SHIP1 to inhibit phosphatidylinositol trikinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. Furthermore, phosphorylated TIGIT recruits SHIP1 via Beta-arrestin 2 and disrupts nuclear factor KB (NF-KB) activation by blocking the autoubiquitination of TNF receptor-associated factor 6 (TRAF6). This series of signaling pathways ultimately suppresses T cell or NK cell function and inhibits cytokine production. PVR is a ligand for both TIGIT and CD226, with a CD226 affinity of 119 nM for PVR. After binding to CD112 or CD155, the intracellular domains of CD226, Ser329 and Tyr322, are phosphorylated. Ser329 phosphorylation promotes the activation of protein kinase (PKC) and the interaction between CD226 and lymphocyte-associated antigen 1 (LFA1). LFA1 is then used for TYN-mediated Tyr322 phosphorylation and CD226-mediated downstream signaling. This series of signal transductions ultimately leads to the activation of T cells or NK cells, promoting cytokine production. TIGIT molecules, present on the surface of T cells or NK cells, also interact with CD226 molecules; TIGIT molecules can directly disrupt the formation of normal CD226 dimers, thereby impairing the normal physiological function of CD226. It can be seen that TIGIT and CD226 are like two ends of a balance scale, subtly regulating the body's immune function through the fulcrum of PVR and the transmission of co-stimulatory and co-inhibitory signals.

Studies on the association between TIGIT and natural killer (NK) cell dysfunction have shown that TIGIT is associated with NK cell exhaustion in tumor-bearing mice and colorectal cancer patients. In several tumor-bearing mouse models, TIGIT blockade can prevent NK cell apoptosis and promote NK cell-dependent tumor immunity. Furthermore, TIGIT blockade in an NK cell-dependent manner leads to effective tumor-specific T cell immunity, enhances the efficacy of anti-PD-L1 antibodies and maintains memory immunity in tumor rechallenge models. This work demonstrates that TIGIT constitutes a previously undervalued immune checkpoint on the surface of NK cells, and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anticancer therapeutic strategy (Zhang, Q., et al. (2018). "Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicitspotent anti-tumor immunity." Nature Immunology.). Since TIGIT is highly expressed on both the surface of T cells and NK cells, while other immune checkpoints such as PD-1 and CTLA-4 are only expressed on the surface of T cells, TIGIT has a greater advantage as a therapeutic target. Therefore, developing antibody drugs targeting this target may have a very bright therapeutic and market prospect.

Summary of the Invention

This invention provides an anti-TIGIT antibody with high affinity for human TIGIT protein. In some embodiments, the anti-TIGIT antibody of this invention is an isolated anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody of this 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 this invention has a high affinity for human TIGIT and possesses suitable ADCC (Antibody-dependent cellular cytotoxicity) activity. In some embodiments, the anti-TIGIT antibody of this invention can be used for therapeutic purposes such as treating various types of cancer and infections, and can also be used for diagnosis and prognosis.

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or fragment thereof specifically binding to an immunoglobulin and an immune receptor tyrosine inhibitory motif T-cell immune receptor (TIGIT) protein, and comprising one, two, three, four, five or all of the following sequences:

(a) VH CDR1, containing the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having a single site of substitution, deletion or insertion;

(b) VH CDR2, containing the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having a single site of substitution, deletion or insertion;

(c) VH CDR3, containing the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence having a single site of substitution, deletion or insertion;

(d) VL CDR1, containing the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence with a single site of substitution, deletion or insertion;

(e)VLCDR2, containing an amino acid sequence as shown in SEQ ID NO: 26 or 43, or an amino acid sequence with a single site of substitution, deletion or insertion;

(f) VL CDR3, containing the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having a single site of substitution, deletion or insertion.

In some embodiments, the present invention provides an isolated antibody or fragment thereof, said antibody or fragment thereof specifically binding to a T-cell immune receptor (TIGIT) protein containing an immunoglobulin and an immune receptor tyrosine inhibitory motif, and comprising:

(a) VH CDR1, containing the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having a single site of substitution, deletion or insertion;

(b) VH CDR2, containing the amino acid sequence shown in SEQ ID NO: 22, or an amino acid sequence having a single site of substitution, deletion or insertion;

(c) VH CDR3, containing the amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence having a single site of substitution, deletion or insertion;

(d) VL CDR1, containing the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence with a single site of substitution, deletion or insertion;

(e) VL CDR2, containing the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having a single-site substitution, deletion, or insertion; and

(f) VL CDR3, containing the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having a single site of substitution, deletion or insertion.

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or fragment thereof specifically binding to an immunoglobulin and an immune receptor tyrosine inhibitory motif T-cell immune receptor (TIGIT) protein, said antibody or fragment thereof comprising one, two, three, four, five or all of the following: VH CDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 22, VH CDR3 as shown in SEQ ID NO: 23, VL CDR1 as shown in SEQ ID NO: 25, VL CDR2 as shown in SEQ ID NO: 26 or 43 and VL CDR3 as shown in SEQ ID NO: 27.

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or fragment thereof specifically binding to an immunoglobulin and an immunoreceptor tyrosine inhibitory motif of a T-cell immune receptor (TIGIT) protein, said antibody or fragment thereof comprising VHCDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 22, VH CDR3 as shown in SEQ ID NO: 23, VL CDR1 as shown in SEQ ID NO: 25, VL CDR2 as shown in SEQ ID NO: 26 or 43, and VL CDR3 as shown in SEQ ID NO: 27.

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or fragment thereof specifically binding to an immunoglobulin and an immunoreceptor tyrosine inhibitory motif of a T cell immune receptor (TIGIT) protein, said antibody or fragment thereof comprising VHCDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 22, VH CDR3 as shown in SEQ ID NO: 23, VL CDR1 as shown in SEQ ID NO: 25, VLCDR2 as shown in SEQ ID NO: 26, and VL CDR3 as shown in SEQ ID NO: 27.

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof, said antibody or fragment thereof specifically binding to an immunoglobulin and an immunoreceptor tyrosine inhibitory motif of a T-cell immune receptor (TIGIT) protein, said antibody or fragment thereof comprising VH CDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 22, VH CDR3 as shown in SEQ ID NO: 23, VL CDR1 as shown in SEQ ID NO: 25, VLCDR2 as shown in SEQ ID NO: 43, and VL CDR3 as shown in SEQ ID NO: 27.

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 κ or λ type. In some embodiments, the light chain constant region is a κ or λ chain constant region. In some embodiments, the light chain constant region is a κ chain constant region. In some embodiments, the light chain variable region is a κ or λ chain variable region. In some embodiments, the light chain variable region is a κ chain variable region. In some embodiments, the antibody or fragment thereof is an isotype of 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.

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 amino acid at position 356 (Eu number) of the heavy chain in the human Fc region is E. In some embodiments, the amino acid at position 386 (Eu number) of the heavy chain in the human Fc region is M. In some embodiments, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or 29. In some embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 30.

In some embodiments, the antibody or fragment thereof includes a 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 homology with 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 includes a 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 homology with an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 34, 36, 38, 40, and 42.

In some embodiments, the antibody or fragment thereof includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 35 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 36.

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.

In some embodiments, the antibody or fragment thereof has an affinity _KD ≤ 100 pM for human TIGIT. In some embodiments, the antibody or fragment thereof has an affinity _KD ≤ 5 nM for human TIGIT.

In some embodiments, the antibody or fragment thereof has ADCC activity. In some embodiments, the antibody or fragment thereof binds to fucose. In some embodiments, the antibody or fragment thereof does not bind to fucose.

On the other hand, the present invention also describes a bispecific antibody, characterized in that the bispecific antibody comprises the antibody fragment described in the present invention and a second antigen-binding fragment that is specific to molecules on immune cells. The molecules include 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.

On the other hand, the present invention also provides compositions comprising the antibodies or fragments thereof disclosed herein, wherein the compositions further comprise pharmaceutically acceptable carriers. In some embodiments, the composition contains less than or equal to 50% of the antibody or fragment thereof bound to fucose. In some embodiments, the composition contains less than or equal to 30% of the antibody or fragment thereof bound to fucose. In some embodiments, the composition contains less than or equal to 20% of the antibody or fragment thereof bound to fucose. In some embodiments, the composition contains less than or equal to 10% of the antibody or fragment thereof bound to fucose. In some embodiments, the composition contains less than or equal to 5% of the antibody or fragment thereof bound to fucose. In some embodiments, the composition contains less than or equal to 1% of the antibody or fragment thereof bound to fucose.

On the other hand, the present invention also provides a polynucleotide that can encode the antibody or a fragment thereof. In some embodiments, the sequence of the polynucleotide encoding the antibody heavy chain is shown in SEQ ID NO: 46; in some embodiments, the sequence of the polynucleotide encoding the antibody light chain is shown in SEQ ID NO: 47.

On the other hand, the present invention also provides a cell comprising one or more polynucleotides encoding an antibody or a fragment thereof disclosed herein.

On the other hand, the present invention also provides a method for preparing the antibody or a fragment thereof, characterized in that cells containing polynucleotides encoding the antibody or a fragment thereof are cultured to express the antibody or the fragment thereof. In some embodiments, the cells are CHO cells, HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, or mouse L cells.

On the other hand, the present invention also provides treatment methods and uses. In some embodiments, the present invention provides a method for treating cancer or infection in a patient in need, comprising administering to the patient an effective dose of an antibody or fragment thereof disclosed in the present invention. In some embodiments, 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, stomach cancer, esophageal cancer, ovarian cancer, kidney 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, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer. In some embodiments, the method further includes administering a second cancer treatment agent to the patient. In some embodiments, the infection is a viral infection, bacterial infection, fungal infection, or parasitic infection.

In some embodiments, the present invention provides a method for treating cancer or infection in a patient in need, comprising: (a) treating cells in vitro with an antibody or fragment thereof disclosed herein; and (b) administering the treated cells to a patient. In some embodiments, the method further comprises isolating cells from an individual prior to step (a). In some embodiments, cells are isolated from a patient. In some embodiments, cells are isolated from a donor individual distinct 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 combinations thereof.

In some embodiments, the present invention also provides the use of anti-TIGIT antibodies or fragments thereof in the preparation of medicaments for treating cancer or infection in patients in need. 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, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. In some embodiments, the invention further includes administering a second cancer treatment agent to the patient. In some embodiments, the infection is a viral infection, bacterial infection, fungal infection, or parasitic infection.

In some embodiments, the present invention also provides the use of cells in the preparation of medicaments for treating cancer or infection, the use comprising: (a) treating the cells in vitro with an antibody or a fragment thereof, 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 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 combinations thereof.

On the other hand, the present invention also provides diagnostic methods and uses. In some embodiments, the present invention provides a method for detecting TIGIT expression in a sample, comprising contacting the sample with an antibody or a fragment thereof under certain conditions, such that the antibody or the fragment thereof binds to TIGIT, and detecting the binding, i.e., TIGIT expression in the sample. In some embodiments, the sample includes tumor cells, tumor tissue, infected tissue, or blood samples.

Attached Figure Description

To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments recorded in this application, wherein:

Figure 1: Schematic diagram of PVR-FC fusion protein construction.

Figure 2: Schematic diagram of TIGIT-FC fusion protein construction.

Figure 3: Results of m10D8 antibody and human TIGIT ELISA experiment.

Figure 4: Results of the m10D8 antibody and monkey TIGIT ELISA experiment.

Figure 5: Results of the m10D8 and mouse TIGIT ELISA experiments.

Figure 6: Results of mouse anti-human TIGIT blocking TIGIT-PVR binding experiment, 10A7 represents m10D8, and BAT5906, an irrelevant antibody, is used as a negative control.

Figure 7: Results of biological activity assay of mouse anti-human TIGIT antibody, m10D8 is represented; BAT5906 is represented as an irrelevant antibody and served as a negative control; Tiragolumab is represented as a positive control.

Figure 8: Comparison of binding capabilities of h10D8V1, h10D8V2, and h10D8V3 with TIGIT. 'A' represents h10D8V1, 'B' represents h10D8V2, and 'C' represents h10D8V3.

Figure 9: Determination of equilibrium dissociation constants of h10D8V1, h10D8V2, h10D8V3 and TIGIT-his tag.

Figure 10: Comparison of biological activities of h10D8V1, h10D8V2, and h10D8V3, where h10D8V1 represents h10D8V2 and h10D8V3 respectively.

Figure 11: Comparison of binding ability of h10D8OF, h10D8V4, and h10D8V5 with TIGIT. * indicates h10D8OF, * indicates h10D8V4, and * indicates h10D8V5.

Figure 12: Comparison of binding ability of h10D8OF, h10D8V2 with TIGIT, where Tiragolumab represents Tiragolumab, h10D8V2 represents h10D8OF.

Figure 13: Detection results of equilibrium dissociation constants of h10D8OF, h10D8V2 and TIGIT-his tag antigens.

Figure 14: Comparison of biological activities of h10D8OF, h10D8V4, and h10D8V5, where h10D8OF, h10D8V4, h10D8V5, and Tiragolumab are represented.

Figure 15: Experimental results of blocking TIGIT and PVR binding by h10D8OF, h10D8V4, and h10D8V5. Tiragolumab is represented, h10D8OF is represented, * represents h10D8V4, h10D8V5 is represented, and BAT5906 is the irrelevant control.

Figure 16: Comparison of the ability of h10D8OF, h10D8V4, and h10D8V5 to block the binding of PVR to TIGIT on the surface of Jurkat cell membrane. h10D8V4 represents h10D8V4, h10D8V5 represents h10D8OF, and BAT5906 represents the irrelevant control.

Figure 17: Comparison of the ability of h10D8OF and Tiragolumab to block the binding of PVR and TIGIT on the surface of Jurkat cell membrane. Tiragolumab represents the control group, h10D8OF represents the control group, and BAT5906 represents the control group.

Figure 18: h10D8OF ADCC activity assay results, representing h10D8OF, Tiragolumab, and irrelevant control BAT5906.

Figure 19: Comparison of ADCC activities between h10D8OF and h10D8OFKF.

Figure 20: Relative induction fold of IFN-γ, 10 represents 10 μg/ml, 25 represents 25 μg/ml.

Figure 21: IL-2 relative induction fold, 10 represents 10 μg/ml, 25 represents 25 μg/ml.

Figure 22: Results of the binding experiment between anti-TIGIT antibody and hTIGIT on the surface of B-hPD-1/hTIGIT dual-humanized mouse T cells. The humanized antibody secondary antibody was anti-human Fc, FITC; the mouse antibody secondary antibody was anti-mouse IgG, Alexa Fluor 647.

Figure 23: Comparison of the binding ability of h10D8OF and Tiragolumab to TIGIT on the cell surface of Jurkat-hTIGIT, where h10D8OF represents h10D8OF and Tiragolumab represents Tiragolumab.

Figure 24: Efficacy of murine antibody monotherapy, where saline represents vehicle, Pembrolizumab monotherapy (0.1 mg/kg) represents Tiragolumab monotherapy (20 mg/kg) represents m10D8.

Figure 25: Efficacy of combined humanized antibody therapy. Saline represents pembrolizumab monotherapy (0.1 mg/kg), h10D8V2 monotherapy (20 mg/kg), Tiragolumab monotherapy (20 mg/kg), and * represents h10D8OF monotherapy (20 mg/kg).

Figure 26: Efficacy of combined humanized antibody therapy. * indicates saline solution, * indicates pembrolizumab, * indicates combined use of h10D8V2 (20 mg/kg) and pembrolizumab (0.1 mg/kg), * indicates combined use of tiragolumab (20 mg/kg) and pembrolizumab (0.1 mg/kg), * indicates combined use of h10D8OF (20 mg/kg) and pembrolizumab (0.1 mg/kg).

Figure 27: Proportion of mCD45 viable cells among MC38 tumor-infiltrating lymphocytes. ∠ represents physiological saline, ∠ represents h10D8OF.

Figure 28: The proportion of CD4+ T cells to mCD45 viable cells. ∠ represents physiological saline, ∠ represents h10D8OF.

Figure 29: The proportion of CD8+ T cells to mCD45 viable cells. ∠ represents physiological saline, ∠ represents h10D8OF.

Figure 30: Proportion of TIGIT+CD4+ T cells among live CD4+ T cells. * indicates physiological saline, * indicates h10D8OF. Figure 31: Proportion of TIGIT+CD8+ T cells among live CD8+ T cells. * indicates physiological saline, * indicates h10D8OF. Figure 32: In vivo pharmacodynamic results. * indicates physiological saline, * indicates mTiragolumab (30 mg/kg), * indicates Atezolizumab (1 mg/kg), * indicates mh10D8OF (10 mg/kg), * indicates mh10D8OF (30 mg/kg), * indicates combination therapy, mh10D8OF (30 mg/kg) + Atezolizumab (1 mg/kg).

Detailed Implementation

definition

It should be noted that the term “a” entity refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

An amino acid is an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by nucleic acids directly or in its precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural 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).

"Conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially change the functional properties of a protein. Examples of amino acid classes containing chemically similar side chains 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; 6) Acidic side chains: aspartic acid and glutamic acid.

In this invention, the term "polypeptide" is intended to cover both the singular and plural forms of "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains composed of two or more amino acids and does not imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of any of the foregoing terms or used interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence. They can be produced in any manner, including chemical synthesis.

In this invention, the term "isolated" as used with respect to antibodies, cells, and nucleic acids, such as "isolated" DNA or RNA, refers to molecules isolated from other DNA or RNA present in the natural source of macromolecules. The term "isolated" as used in this invention also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or cell culture medium, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring and will not exist in their natural state. The term "isolated" is also used in this invention to refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides.

In this invention, the term "recombination" refers to polypeptides or polynucleotides, meaning polypeptides or polynucleotides that do not exist naturally. Unlimited embodiments can generate polynucleotides or polypeptides that do not normally exist by combination.

"Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing comparable positions in each sequence. Molecules are homologous at those positions when the positions in the compared sequences are occupied by the same bases or amino acids. The degree of homology between sequences is a function of the number of shared matching or homologous positions. The terms "irrelevant" or "non-homologous" sequences indicate less than 40% identity with one of the sequences disclosed in this invention, such as less than 25%.

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

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that shares a degree of homology or sequence identity with the nucleotide sequence of a nucleic acid or its complement. Homologous nucleic acids of double-stranded nucleic acids are defined as nucleic acids having a nucleotide sequence that shares a degree of homology with its or its complement's sequence. On the one hand, homologous nucleic acids are capable of hybridizing with nucleic acids or their complements. Similarly, "equivalent polypeptide" refers to a polypeptide that shares a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some respects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some respects, the equivalent polypeptide or polynucleotide has 1, 2, 3, 4, or 5 additions, deletions, substitutions, or combinations thereof compared to the reference polypeptide or polynucleotide. In some respects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

Hybridization reactions can be performed under various “strictness” conditions. Low-strictness hybridization reactions are typically performed at approximately 40°C in solutions of approximately 10 × SSC or equivalent ionic strength/temperature. Moderate-strictness hybridization reactions are typically performed at approximately 50°C in solutions of approximately 6 × SSC, and highly strictness hybridization reactions are typically performed at approximately 60°C in solutions of approximately 1 × SSC. Hybridization reactions can also be performed under “physiological conditions” well known to those skilled in the art. Examples of unrestricted physiological conditions are those generally referring to temperature, ionic strength, pH, and ^Mg²⁺ concentration as typically present in cells.

Polynucleotides consist of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and when the polynucleotide is RNA, thymine is replaced by uracil (U). Therefore, the term "polynucleotide sequence" is a letter representation of the polynucleotide molecule. This letter representation can be entered into a database in a computer with 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 a portion thereof; a portion of a gene having at least two different forms (i.e., two different nucleotide sequences) is called a "polymorphic region of the gene." A polymorphic region can be a single nucleotide, exhibiting different identities in different alleles.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Examples of unrestricted polynucleotides include: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modification is present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of the nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the polynucleotide of this disclosure includes a double-stranded form and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

When the term "coding" is applied to polynucleotides, it refers to a polynucleotide called the "coding" polypeptide, which, if in its native state or when manipulated by methods known to those skilled in the art, can be transcribed and/or translated to produce mRNA of the polypeptide and/or fragments thereof. The antisense strand is the complementary sequence of such nucleic acid from which the coding sequence can be deduced.

In this invention, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. This embodiment, including but not limited to, includes complementarity-determining regions (CDRs), variable regions (VH) or variable regions (VL) of the heavy or light chain or their ligand-binding moieties, heavy chain constant regions (CH) or constant regions (CL) of the light chain, framework (FR) regions, or any portion thereof, or at least a portion of the binding protein. CDR regions include the CDR region of the light chain (LCDR) and the CDR region of the heavy chain (HCDR).

In this invention, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some respects, these regions are linked by short linker peptides of 10 to approximately 25 amino acids. The linker may be enriched with glycine to increase flexibility, and with serine or threonine to increase solubility, and may link the N-terminus of the VH to the C-terminus of the VL, and vice versa. Although the constant region of the protein is removed and the linker is introduced, it retains the specificity of the original immunoglobulin. ScFv molecules known in the art are described, for example, in U.S. Patent 5,892,019.

The term "antibody" encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chain classes include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with further subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and their conferred functional specificities are known. Each variation of these types and isotypes is readily apparent to those skilled in the art and is therefore within the scope of this invention; all immunoglobulin types are clearly within the scope of this invention. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 Daltons. These four chains are typically connected by disulfide bonds in a “Y” configuration, with the light chain starting from the “Y” opening and continuing to surround the heavy chain through a variable region.

The antibodies, antigen-binding fragments, or derivatives disclosed in this invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab′, and F(ab′) ₂ , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies of the LIGHT antibody disclosed in this invention). The immunoglobulin or antibody molecules disclosed in this invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulins.

Light chains can be divided into kappa or lambda (κ, λ). Each heavy chain can bind to either a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are linked by covalent bonds, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration forked end to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ; the variable region of the immunoglobulin λ light chain is Vλ.

Both the 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 chain (VL) and heavy chain (VH) determine antigen recognition and specificity. Conversely, the constant regions of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant regions increases as they become more distant from the antigen-binding site or amino terminus of the antibody. The N-terminal region is the variable region, and the C-terminal region is the constant region; the CH3 and CL domains contain the carboxyl termini of the heavy and light chains, respectively.

As described above, the variable region enables antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VL and VH domains, or complementarity-determining regions (CDRs), binds to form a variable region that defines a three-dimensional antigen-binding site. This antibody quaternary structure forms antigen-binding sites at the ends of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three CDRs in each of the VH and VL chains (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3). In some cases, such as certain immunoglobulin molecules derived from camel-dwelling animals or modified immunoglobulins based on camel-dwelling immunoglobulins, the complete immunoglobulin molecule may consist only of the heavy chain, without the light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, assuming the antibody presents its three-dimensional conformation in an aqueous environment, six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are specifically positioned to form short, discontinuous amino acid sequences that form the antigen-binding domain. The remaining amino acids in the framework region, known as the "framework region," exhibit less intermolecular variability. The framework region largely adopts a β-sheet conformation, with CDRs forming linked loop structures or, in some cases, forming part of a β-sheet. Thus, the framework region positions the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions. The antigen-binding domain formed by a CDR at a specific location defines a surface complementary to an epitope on an immunoreactive antigen, which facilitates non-covalent binding of the antibody to its homologous epitope. For any given heavy or light chain variable region, an amino acid containing the CDR and framework region can be identified by a person skilled in the art according to common definitions (see, for example, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983) and Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)).

According to the definition of CDRs by Kabat and Chothia, they include overlaps or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to a CDR of an antibody or its antigen-binding fragment is within the scope of the terminology defined and used in this invention. The exact residue numbering containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues contain a particular CDR based on the amino acid sequence of the variable region of the antibody.

Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can readily apply this "Kabat numbering" system to any variable region sequence without relying on any experimental data outside the sequence itself. The "Kabat numbering" used in this invention refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983).

The Kabat numbering system describes the CDR regions as follows: HCDR1 begins at approximately the 31st amino acid (approximately 9 residues after the first cysteine residue), consists of approximately 5–7 amino acids, and ends at the next tryptophan residue. HCDR2 begins at the 15th residue after the end of HCDR1, consists of approximately 16–19 amino acid residues, and ends at the next arginine or lysine residue. HCDR3 begins at approximately the 33rd amino acid residue after the end of HCDR2; consists of 3–25 amino acids; and ends with the sequence W-G-X-G, where X represents any amino acid. LCDR1 begins at approximately the 24th residue (after the cysteine residue); consists of approximately 10–17 residues; and ends at the next tryptophan residue. LCDR2 begins at approximately the 16th residue after the end of LCDR1, and consists of approximately 7 residues. LCDR3 begins at approximately the 33rd residue after LCDR2 (i.e., after the cysteine residue); it comprises approximately 7-11 residues and ends with the sequence F or W-G-X-G, where X refers to any amino acid.

The antibodies disclosed in this invention can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

"Light chain-heavy chain pair" refers to a set of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

As described above, the subunit structures and three-dimensional configurations of constant regions of various immunoglobulin species are well known. In this invention, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (mostly N-terminal) constant region of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is connected to the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

In this invention, the term "CH2 domain" includes, when using conventional numbering schemes, the portion of the heavy chain molecule extending from approximately residue 244 to residue 360 of the antibody (residues 244 to 360, Kabat numbering system; residues 231–340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983)). The CH2 domain is unique because it does not pair tightly with other domains, but rather inserts two N-linked branched carbohydrate chains between the two CH2 domains of the intact native IgG molecule. It has also been documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, containing approximately 108 residues.

In this invention, the term "hinge region" includes the heavy chain portion connecting the CH1 and CH2 domains. The hinge region contains approximately 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 domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

In this invention, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. Cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, with the two heavy chains linked by two disulfide bonds at positions 239 and 242 (positions 226 or 229 in the EU numbering system) in the Kabat numbering system.

In this invention, the term "chimeric antibody" is considered to refer to any antibody whose immunoreactive region or site is derived from a first species, while its constant region (which may be whole, partial, or modified in this invention) is derived from a second species. In some embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate), while the constant region is human.

"Specific binding" generally refers to the binding of an antibody to an antigenic epitope via its antigen-binding domain, and this binding requires complementarity between the antigen-binding domain and the epitope. According to this definition, when an antibody binds to an epitope more readily through its antigen-binding domain than to a random, unrelated epitope, it is said to have "specifically bound" that epitope. The term "specific" is used in this invention to define the relative affinity of a particular antibody for a particular epitope.

In this invention, the term "treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing or slowing (reducing) adverse physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: relief or elimination of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction or elimination (whether partial or complete). "Treatment" also means an extension of life compared to the expected lifespan without treatment. Those in need of treatment include those who already have the condition or disorder, those who are susceptible to the condition or disorder, or those who need to prevent the condition or disorder.

The terms "subject," "individual," "animal," "patient," or "mammal" generally refer to any subject requiring diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc.

In this invention, phrases such as “patient in need of treatment” or “subject in need of treatment” include subjects who benefit from the administration of the antibodies or compositions disclosed in this invention for detection, diagnostic procedures and/or treatment, such as mammalian subjects.

Anti-TIGIT antibody

This invention provides anti-TIGIT antibodies with high affinity for 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 for human TIGIT and suitable ADCC activity.

Therefore, the present invention provides an anti-TIGIT antibody or a fragment thereof, which can specifically bind to the TIGIT protein.

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 demonstrated in the experiments in the examples, antibodies containing these CDR regions, whether mouse or humanized, exhibit effective binding and inhibitory activity against TIGIT.

In some embodiments, the anti-TIGIT antibody disclosed in this invention comprises a VH as shown 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 with each of them, or a VL as shown 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 with each of them. In some embodiments, the VH having an overall sequence identity of 80%, 85%, 90%, 95%, 98%, or 99% retains the CDR (SEQ ID NO: 21-23, 25-27, 43, or an amino acid sequence thereof with a single site of substitution, deletion, or insertion). In one embodiment, the VH has an amino acid sequence as shown in SEQ ID NO: 35, and the VL has an amino acid sequence as shown in SEQ ID NO: 36.

In some embodiments, the anti-TIGIT antibody disclosed in this invention comprises a human Fc region. The constant region sequences of the heavy and light chains may, for example, be SEQ ID NO: 28 (or 29) and 30. Compared to SEQ ID NO: 28, in SEQ ID NO: 29, amino acid 239 is represented by an E instead of D (based on Eu encoding, position 356; Kabat encoding, position 377), and amino acid 241 is represented by an M instead of L (based on Eu encoding, position 358; Kabat encoding, position 381).

In some embodiments, the heavy chain sequence of the anti-TIGTI antibody disclosed in this invention is shown in SEQ ID NO: 44; the light chain sequence is shown in SEQ ID NO: 45.

Those skilled in the art should also understand that the antibodies disclosed in this invention can be modified, and their amino acid sequences after modification differ from the amino acid sequences of the naturally occurring binding polypeptides from which the antibody is derived. For example, the polypeptide or amino acid sequence derived from the same specified protein can be similar to the starting sequence, such as having a certain percentage of identity with the starting sequence, for example, its identity with the starting sequence can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

In some embodiments, the antibody comprises an amino acid sequence or one or more groups that are generally unrelated to the antibody. Representative modifications are described in more detail below. For example, the antibodies disclosed in this invention may comprise a resilient linker sequence or may be modified to add functional groups (e.g., PEG, drugs, toxins, or tags).

In some embodiments, the antibody or fragment thereof does not bind fucose. In some embodiments, the antibody or fragment thereof has up to three (or up to two, or one) amino acid residues modified with fucose. In some embodiments, the formulation containing the antibody or fragment thereof contains up to less than 0.01%, 0.1%, 1%, 2%, 3%, 4%, or 5% of the protein molecule modified with fucose. In some embodiments, antibodies with reduced or removed fucose modification have stronger ADCC and are suitable for certain therapeutic uses.

The antibodies, variants, or derivatives disclosed in this invention include modified derivatives, i.e., modified by covalent linking any type of molecule to the antibody, wherein the covalent linking does not prevent the antibody from binding to the epitope. Examples include, but are not limited to, modifications such as glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization via known protecting/blocking groups, proteolytic cleavage, and linking to cellular ligands or other proteins. Any of these numerous chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, antibodies may contain one or more non-classical amino acids.

In some implementations, antibodies may be conjugated with therapeutic agents, drug precursors, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG.

Antibodies can be conjugated or fused with therapeutic agents, which may include detectable markers such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutic agents or diagnostic agents, cytotoxic agents that may be drugs or toxins, ultrasound enhancers, non-radioactive markers and combinations thereof, and other such agents known in the art.

Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

Bifunctional molecules

TIGIT is an immune receptor and also a tumor antigen. As a tumor antigen targeting molecule, antibodies or antigen-binding fragments that specifically bind to TIGIT can be combined with another antigen-binding fragment that has immune cell specificity to produce bispecific antibodies.

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. Targeting molecules on the immune cells include, for example, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptors (KIRs), and CD47. Specific examples of bispecific antibodies include, but are not limited to, TIGIT/PD-1, TIGIT/LAG3, and TIGIT/CD47. As illustrated in the examples, the TIGIT antibody and PD-L1 antibody of the present invention exhibit a synergistic effect.

In some implementations, antibodies or antigen-binding fragments that specifically bind to TIGIT can be combined with another antigen-binding fragment that has tumor antigen specificity to produce bispecific antibodies. A "tumor antigen" is an antigenic substance produced by tumor cells that triggers an immune response in the host. Tumor antigens can be used to identify tumor cells and can serve as candidate drugs for cancer treatment. Normal proteins in the body are not antigenic, but certain proteins are produced or overexpressed during tumorigenesis, and are therefore "foreign" to the body. These proteins may include normal proteins that are well isolated by the immune system, proteins that are usually produced in very small amounts, proteins that are usually produced only at certain stages of development, or proteins whose structure has been modified due to mutations.

Numerous tumor antigens have been discovered in this field, and novel 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, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and cytokinins.

In some respects, the monovalent unit is specific for proteins overexpressed on tumor cells compared to corresponding non-tumor cells. "Corresponding non-tumor cells" as used here refers to non-tumor cells of the same cell type of origin as tumor cells. It is worth noting that such proteins are not necessarily different from tumor antigens; non-limiting examples include: carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectal, breast, lung, pancreatic, and gastrointestinal cancers; neuroregulatory protein receptors (HER-2, neu, or c-erbB-2), which are frequently overexpressed in breast, ovarian, colon, lung, prostate, and cervical cancers; epidermal growth factor receptor (EGFR), which is highly expressed in a range of solid tumors including breast, head and neck, non-small cell lung cancer, and prostate cancer; and desialic acid. Glycoprotein receptors; transferrin receptors; serine protease inhibitor complex receptors, expressed on hepatocytes; fibroblast growth factor receptors (FGFRs), overexpressed on pancreatic ductal adenocarcinoma cells; vascular endothelial growth factor receptors (VEGFRs) for anti-angiogenic gene therapy; folate receptors, selectively overexpressed in 90% of non-adhesive ovarian cancers; cell surface glycocalyxes; carbohydrate receptors; and polymerase receptors, which primarily deliver genes to respiratory epithelial cells and are potentially attractive for treating conditions such as cystic fibrosis. Non-limiting examples of bispecificity in this area include: TIGIT/EGFR, TIGIT/Her2, TIGIT/CD33, TIGIT/CD133, TIGIT/CEA, and TIGIT/VEGF.

This invention also discloses different forms of bispecific antibodies. In some embodiments, the anti-TIGIT fragment and another fragment are selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies, respectively. In some embodiments, the bispecific antibody further includes an Fc fragment.

This invention also 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 as described in this invention can be selectively linked to immune cytokines or ligands via peptide linkers. The linked immune cytokines 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 immune checkpoint blockade effects with local immune modulation at the tumor site.

Polynucleotides encoding antibodies and methods for preparing antibodies

This invention also discloses isolated polynucleotides or nucleic acid molecules encoding the antibodies described herein and their antigen-binding fragments or derivatives. The polynucleotides disclosed herein can encode the entire heavy and light chain variable regions of the aforementioned antibodies or antigen-binding fragments or derivatives. Furthermore, the polynucleotides disclosed herein can encode portions of the heavy and light chain variable regions of the aforementioned antibodies or antigen-binding fragments or derivatives.

In some embodiments, the polynucleotide sequence encoding the anti-TIGIT antibody h10D8OF is shown in SEQ ID NO: 46 and 47; wherein sequence SEQ ID NO: 46 encodes the heavy chain that produces the anti-TIGIT antibody h10D8OF; and sequence SEQ ID NO: 47 encodes the light chain that produces the anti-TIGIT antibody h10D8OF.

The sequence of SEQ ID NO: 46 is as follows:

The sequence of SEQ ID NO: 47 is as follows:

In some embodiments, hybridoma technology is employed to prepare the anti-TIGIT antibody or its antigen-binding fragment of the present invention. Monoclonal antibodies, such as those described in Kohler and Milstein, Nature, 256:495 (1975), are prepared using, for example, hybridoma methods. In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce lymphocyte production or the production of antibodies that specifically bind to the immunizing agent.

Immunoassays typically include protein antigens, fragments thereof, or fusion proteins thereof. Peripheral blood lymphocytes are typically used if human cells are desired, and splenic lymphocytes or lymph node cells are used if non-human mammalian cells are desired. In some embodiments, splenic lymphocytes are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are typically used. In some embodiments, splenic lymphocytes and mouse myeloma cells are fused. The hybridoma cells are cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymine (“HAT medium”), substances that prevent the growth of HGPRT-deficient cells. In some embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to target antigens.

After identifying the desired hybridoma cells, the clone can be subcloned using a limiting dilution step and grown using standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco modified Eagle medium and RPMI-1640 medium.

In some implementations, monoclonal antibodies secreted by subclones can be isolated or purified using conventional techniques, such as protein A-agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be prepared using 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 using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells described herein serve as a preferred source of such DNA. DNA encoding the antibodies described herein can also be synthesized according to antibody sequences using conventional methods. The isolated or synthesized DNA is placed into an expression vector and then transfected into host cells such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, ape COS cells, PER.NS0 cells, SP2/0, YB2/0, or myeloma cells that do not additionally produce immunoglobulins, thereby obtaining synthetic monoclonal antibodies in recombinant host cells.

In some embodiments, hybridoma technology is used to prepare the anti-TIGIT antibody or its antigen-binding fragment of the present invention. Mice are immunized with a fusion protein containing the TIGIT extracellular region. After immunization, splenic lymphocytes from the mice are fused with myeloma cells. The hybridomas are screened using the fusion protein containing the TIGIT extracellular region, and positive hybridomas are subjected to limiting dilution and further subcloning. The binding ability of the hybridoma strains to the fusion protein containing the TIGIT extracellular region is reassessed to prepare the anti-TIGIT antibody. In some embodiments, the fusion protein containing the TIGIT extracellular region is TIGIT-Fc. In some embodiments, the mice are 6-8 week old female Balb/c mice.

Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, the variable and constant regions of the antibodies or antigen-binding fragments disclosed herein are fully human. Fully human antibodies can be prepared using techniques disclosed in the art and techniques described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are found in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, the entire contents of which are incorporated herein by reference.

In some embodiments, the prepared antibodies do not elicit a harmful 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 using techniques recognized in the art to reduce their immunogenicity. For example, the antibodies can be humanized, primate-like, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but have lower immunogenicity in humans. This can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementarity-determining regions (CDRs) into the human framework and constant regions, with or without key framework residues; or (c) transplanting the entire non-human variable region but “hiding” them by replacing surface residues with human-like portions.

Deimmunization can also be used to reduce the immunogenicity of antibodies. “Deimmunization” involves altering antibodies to modify T-cell epitopes (see, for example, International Application Publications: WO/9852976A1 and WO/0034317A2). For example, sequences of the variable heavy and light chains from the starting antibody are analyzed, generating a “map” of human T-cell epitopes from each V region, showing the position of the epitope relative to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T-cell epitopes from the T-cell epitope map are analyzed to identify alternative amino acid substitutions with a lower risk of altering the final antibody activity. A series of optional variable heavy and light chain sequences containing combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding peptides. Typically, 12 to 24 antibody variants are generated, and their binding capacity and/or function are tested. Genes containing the modified variable regions and human constant regions of the complete heavy and light chains are then cloned into expression vectors, and the plasmids are subsequently transfected into cell lines to produce complete antibodies. Then, by comparing antibodies in appropriate biochemical and biological experiments, the best variant can be identified.

The binding specificity of the antibody or antigen-binding fragment disclosed in this invention can be detected by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

Examples of techniques that can be used to produce single-chain Fv (scFv) 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.

For certain applications, including the use of antibodies in humans and in vitro assays, chimeric antibodies, humanized antibodies, or fully human antibodies may be preferred. Chimeric antibodies are a class of molecules in which different parts of an antibody are derived from different animal species, such as antibodies having the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gilles 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.

Humanized antibodies are antibody molecules derived from non-human antibodies capable of binding to a target antigen having one or more non-human complementarity-determining regions (CDRs) and a framework region derived from a human immunoglobulin molecule. Typically, certain framework residues in the human framework region are replaced by corresponding residues from a CDR donor antibody, preferably residues that improve antigen binding. These framework substitutions can be identified using methods known in the art, such as by mimicking the interaction between the CDR and framework residues to identify framework residues that play an important role in antigen binding and by sequence comparison to identify aberrant framework residues at specific positions. (Refer to Queen et al., U.S. Pat. No. 5, 585, 089; Riechmann et al., Nature 332: 323 (1988), the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, such as CDR transplantation (EP239, 400, PCT publication WO 91/09967, U.S. 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 2). 8(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 (U.S. Pat. No. 5, 565, 332), the entire contents of which are incorporated herein by reference.

Fully human antibodies are particularly desirable for treating human patients. Fully human antibodies can be prepared using a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, the entire contents of which are incorporated herein by reference.

Human antibodies can also be produced by transgenic mice that do not express functional endogenous immunoglobulins but do express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be randomly introduced or introduced into mouse embryonic stem cells via homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy chain and light chain immunoglobulin genes can be defunctionalized by homologous recombination, either separately or simultaneously, through the introduction of human immunoglobulin gene loci. In particular, homozygous deletion of the JH region can prevent the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then cultured to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a conventional manner with selected antigens, such as all or part of the desired polypeptide target. Monoclonal antibodies targeting the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by transgenic mice undergo rearrangement during B cell differentiation, followed by class switching and somatic mutations. Therefore, this technology can be used to produce therapeutic IgG, IgA, IgM, and IgE antibodies. A review of this technology for producing fully human antibodies can be found in Lonberg and Huszar Int. Rev. Immunol. 73: 65-93 (1995). For a detailed discussion of this technology for producing fully human antibodies and human monoclonal antibodies, and the steps involved in producing such antibodies, see PCT publications WO 98/24893, WO 96/34096, WO 96/33735, and U.S. Patents 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. In addition, companies such as Abgenix (Freemont, Calif.) and GenPharm (San Jose, Calif.) can use similar technologies to provide fully human antibodies against selected antigens.

A technique known as “guided selection” can also be used to produce fully human antibodies that recognize selective epitopes. In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the screening of fully human antibodies that recognize 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).

In other embodiments, DNA encoding the desired monoclonal antibody can be readily isolated and sequenced using conventional methods, such as oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Isolated and subcloned hybridoma cells are preferred sources of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce immunoglobulins. More specifically, the isolated DNA (which may be synthetic, as described herein) can be used to clone sequences of constant and variable regions for antibody preparation, as described in Newman et al. and U.S. Patent 5,658,570, published January 25, 1995, the entire contents of which are incorporated herein by reference. Essentially, this requires extracting RNA from selected cells and converting it into cDNA, followed by amplification using Ig-specific primers via PCR. Suitable probes for this purpose are also mentioned in U.S. Patent 5,658,570. As will be discussed in more detail below, it is possible to culture transformed cells expressing the desired antibodies in relatively large quantities to meet clinical and commercial needs for immunoglobulins.

Furthermore, using conventional recombinant DNA techniques, one or more CDRs of the antibody or antigen-binding fragment of the present invention can be inserted into the frame region, for example, into a human frame region, to construct a humanized non-fully human antibody. The frame region can be a naturally occurring or common frame region, and preferably a human frame region (see Chothia et al., J. Mol. Biol. 278: 457-479 (1998), which lists a series of human frame regions). Preferably, the polynucleotide generated by combining the frame region and CDR 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 within the frame region, and preferably amino acid substitutions that improve the binding of the antibody to its antigen. Additionally, this method can be used to substitute or delete cysteine residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby producing an antibody molecule lacking one or more interchain disulfide bonds. Other modifications to the polynucleotide within the scope of the art are also covered in this invention.

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)) involves splicing genes from mouse antibody molecules with appropriate antigen specificity and genes from human antibody molecules with appropriate biological activity. In this invention, chimeric antibodies are molecules whose different parts are derived from different animal species, such as those containing a variable region derived from mouse monoclonal antibodies and a constant region of human immunoglobulins.

Furthermore, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10:1455-1460 (1992), specifically, this technique produces primate antibodies containing monkey variable region and human constant region sequences, the entire contents of which are incorporated herein by reference. In addition, this technique is also mentioned 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.

Antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines for antibody production can be selected, constructed, and cultured using techniques well-known to those skilled in the art. 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*, D.L. Hacker, F.M. Wurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including supplementary information, are incorporated herein by reference.

In some embodiments, the DNA encoding the antibody described herein can be designed and synthesized according to the antibody's amino acid sequence using conventional methods, placed into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector 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 for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors 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.

In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes, and after being cultured in a selective medium, the successfully transfected cells grow in large numbers, producing the desired target protein.

Furthermore, mutations can be introduced into the nucleotide sequence encoding the antibody of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis leading to amino acid substitutions and PCR-mediated mutations. Preferably, the variants (including derivatives) encode substitutions of less than 50 amino acids, less than 40 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, less than 5 amino acids, less than 4 amino acids, less than 3 amino acids, or less than 2 amino acids relative to the reference heavy chain variable regions HCDR1, HCDR2, HCDR3 and light chain variable regions LCDR1, LCDR2, or LCDR3. Alternatively, mutations can be randomly introduced along all or part of the coding sequence, for example, through saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

Cancer treatment

In this invention, the antibodies, antigen-binding fragments, or derivatives of this invention can be used in certain therapeutic and diagnostic methods.

This invention will further describe antibody-based therapies in which the antibodies described herein are administered to patients, such as animals, mammals, and humans, to treat one or more disorders or conditions described herein. The therapeutic compounds of this invention include, but are not limited to, the antibodies described herein (including the antigen-binding fragments and derivatives described herein) and nucleic acids or polynucleotides encoding the antibodies described herein (including the antigen-binding fragments and derivatives described herein).

The antibodies described in this invention can also be used to treat or inhibit cancer. TIGIT can be overexpressed in tumor cells. Tumor-derived TIGIT can bind to PD-1 on immune cells, thereby limiting anti-tumor T-cell immunity. Results from the use of small molecule inhibitors or monoclonal antibodies targeting TIGIT in mouse tumor models demonstrate that TIGIT-targeted therapies are an important alternative and realistic approach for effectively controlling tumor growth. As demonstrated in the examples, anti-TIGIT antibodies can activate adaptive immune response mechanisms, thereby improving the survival rate of cancer patients.

Therefore, in some embodiments, methods for treating cancer in patients with this need are provided. In one embodiment, the method involves administering an effective dose of the antibody described in this invention to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient expresses, overexpresses, or is induced to express TIGIT. For example, induced expression of TIGIT can be achieved by administering a tumor vaccine or radiotherapy.

Tumors expressing the TIGIT protein include bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral cancer, colon cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, and small cell lung cancer. Therefore, the antibody disclosed in this invention can be used to treat any one or more of these cancers.

Cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, are also provided in this invention. Suitable cells can be contacted with the anti-TIGIT antibody described herein (or, alternatively, engineered to express the anti-TIGIT antibody described herein). 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. Cells (e.g., T cells) include, but are not limited to, types such as tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, and combinations thereof.

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

The antibodies or antigen-binding fragments or their derivatives described in this invention can 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, such disease disorders including leukemia {including acute leukemia [e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia)] and chronic leukemia [e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia]}, polycythemia vera, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and cancers such as fibrosarcoma, myxosarcoma, and lipoma. Sarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, 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, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, neuroblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

combination therapy

In other embodiments, the compositions of the present invention are administered in combination with antitumor agents, antiviral agents, antibacterial agents, antibiotics, or antifungal agents. Any of these agents known in the art can be administered in the compositions of the present invention.

In other embodiments, the compositions of the present invention are administered in combination with chemotherapeutic agents. Chemotherapeutic agents that can be administered with the compositions of the present invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin D), anti-estrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouracil, interferon α-2b, glutamate, styromycin, mercaptopurine, and 6-thioguanine), and cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estmustine, hydroxyurea, and methylbenzyl). Hydrazine, mitomycin, busulfan, cisplatin and vincristine sulfate), hormones (such as medroxyprogesterone, estradiol sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorestradiol and testrolide), nitrogen mustard derivatives (e.g. melphalan, chlorambucil, dichloromethyldiethylammonium (nitrogen mustard) and thiotepa), steroids and combinations thereof (such as betamethasone sodium phosphate), and other compounds (such as dazometazidine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate and etoposide).

In some embodiments, the compositions of the present invention are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

In some embodiments, the compositions of the present invention are administered in combination with other treatment or preventative measures, such as radiotherapy.

This invention also provides combination therapies, including the use of one or more of the anti-TIGIT antibodies of this invention and a second anticancer agent (chemotherapeutic agent). Examples of chemotherapeutic agents include immunotherapeutic agents, including but not limited to therapeutic antibodies suitable for treating patients. Some examples of therapeutic antibodies include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, and bivazumab. zumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutuzumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, ducigituzumab ( dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromoxicillinab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotuzumab umab), futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab ab), labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumumab ab), necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumo (Mab), pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab Taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and 3F8 are among the drugs used. Rituximab can be used to treat indolent B-cell carcinomas, including marginal zone lymphoma, WM, CLL, and small lymphocytic lymphoma. The combination of rituximab and chemotherapy drugs is particularly effective.

In some embodiments, the compounds and compositions described herein may be used or combined with one or more other therapeutic agents. These one or more therapeutic agents include, but are not limited to, Abl inhibitors, activated CDC kinase (ACK), adenosine A2B receptor (A2B), apoptosis signal-regulated kinase (ASK), Auroa kinase, Bruton's tyrosine kinase (BTK), BET-bromodomain (BRD) such as BRD4, c-Kit, c-Met, CDK-activated kinase (CAK), calmodulin-dependent protein kinase (CaMK), cyclin-dependent kinase (CDK), casein kinase (CK), discoid 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, and lymphocytes. Cell-specific protein tyrosine kinase (LCK), lysyl oxidase protein, lysyl oxidase-like protein (LOXL), LYN, matrix metalloproteinase (MMP), 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 thereof.

In some embodiments, the anti-TIGIT antibody of the present invention can be used in conjunction with an immune checkpoint inhibitor. Immune checkpoints are a class of molecules in the immune system that can be either upregulated (co-stimulatory molecules) or downregulated (co-inhibitory molecules). Many cancers protect themselves from the immune system by suppressing T-cell signaling through agonists of co-inhibitory molecules or antagonists of co-stimulatory molecules. Immune checkpoint agonists or antagonists can help block this protective mechanism. Immune checkpoint agonists or antagonists can 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.

For any of the above combination therapies, anti-TIGIT antibodies can be administered concurrently with or separately from another anticancer agent. When administered alone, anti-TIGIT antibodies can be administered before or after the administration of another anticancer agent.

Treatment of infection

As demonstrated in the experimental examples, the antibodies of the present invention can activate an immune response, thereby being used to treat infections.

Infection is the process by which pathogenic agents invade the tissues of an organism, multiply, and the host tissues respond to these organisms and the toxins they produce. Infections can be caused by infectious agents such as viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas and lice, fungi such as tinea, and other large parasites such as tapeworms and other worms. In some cases, infectious agents are bacteria, such as Gram-negative bacteria. In others, infectious agents are viruses, such as DNA viruses, RNA viruses, and retroviruses. Non-limiting examples of viruses include adenoviruses, Coxsackieviruses, Epstein-Barr virus (EBV), hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, HIV, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, and varicella-zoster virus.

The antibodies of this invention can also be used to treat infectious diseases caused by microorganisms, or to eliminate microorganisms by targeting and binding to microorganisms and killing them with immune cells. In one respect, microorganisms include viruses (RNA and DNA viruses), Gram-positive bacteria, Gram-negative bacteria, protozoa, or fungi. Table 4 provides non-limiting examples of infectious diseases and related microorganisms.

Treatment

The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the specific antibody and its antigen-binding fragments or derivatives used, the patient's age and weight, general health condition, sex and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors will be determined by a healthcare professional, including those skilled in the art. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

Methods of administration of antibodies and their antigen-binding fragments include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral administration. Antibodies or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal membranes (e.g., oral mucosa, rectal and intestinal mucosa), and can be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antibodies of the present invention can be administered orally, rectally, parenterally, intracerebrospinally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), orally, or via oral or nasal spray.

The term "parenteral" as used in this invention refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

The administration method can be systemic or local. Furthermore, it may be necessary to introduce the antibodies of the present invention into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be assisted by connecting an intraventricular catheter to a reservoir such as an Ommaya reservoir. Administration can also be via the lungs, for example, by using an inhaler or nebulizer, and using nebulized formulations.

It may be necessary to apply the antibody peptides or compositions of the present invention topically to the area requiring treatment; this can be achieved, but is not limited to, local infusion during surgery, such as topical application in conjunction with postoperative wound dressings, by injection, through a catheter, by means of suppositories, or by means of implants that are 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 present invention, care must be taken to use materials that do not absorb proteins.

In some embodiments, the compositions of the present invention comprise nucleic acids or polynucleotides encoding proteins, which can be administered in vivo to promote the expression of the proteins encoded therein by constructing them as part of a suitable nucleic acid expression vector, and then the aforementioned partial vector is administered to make it an intracellular part, for example by using a retroviral vector (see U.S. Patent 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., gene gun; Biolistic, DuPont), or by coating with lipids or cell surface receptors or transfection reagents, or by administration by linking to a homeobox peptide known to enter the cell nucleus (see, for example, Joliot et al., 1991, Proc. Natl. A cad. Sci. USA 88: 1864-1868), etc. Optionally, the nucleic acid can be introduced into the cell and integrated into the host cell DNA for expression via homologous recombination.

The dosage of the antibody of this invention can be determined using standard clinical techniques. This dosage will be effective in treating, inhibiting, and preventing inflammation, immune or malignant diseases, disorders, or conditions. Additionally, in vitro experiments can optionally be used to help determine the optimal dosage range. The accurate dosage used in the formulation also depends on the route of administration and the severity of the disease, disorder, or condition, and should be determined based on the physician's judgment and each patient's situation. The effective dosage can be deduced from dose-response curves obtained from in vitro or animal model testing systems.

In some embodiments, the antibody of the present invention is typically administered to a patient at a dose of 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg to 20 mg/kg, or 1 mg/kg to 10 mg/kg. Generally, human antibodies have a longer half-life in the human body than antibodies of other species due to the immune response to exogenous peptides. Therefore, lower doses of human antibodies and less frequent administration are generally feasible. Furthermore, modifications such as lipolysis can enhance antibody uptake and tissue penetration (e.g., into the brain), thereby reducing the dose and frequency of administration of the antibody of the present invention.

Methods for in vitro testing for the treatment of infectious or malignant diseases, disorders, or conditions typically involve administering the antibodies, antigen-binding fragments, or derivatives described in this invention, followed by in vivo testing of the desired therapeutic or prophylactic activity in an acceptable animal model, and finally administration to humans. Suitable animal models (including transgenic animals) are well known to those skilled in the art. For example, in vitro assays demonstrating the therapeutic use of the antibodies or antigen-binding fragments described in this invention include the effect of the antibody or antigen-binding fragment on a cell line or patient tissue sample. The effect of the antibody or antigen-binding fragment on the cell line and/or tissue sample can be detected using techniques known to those skilled in the art, such as those disclosed in other parts of this invention. According to the present invention, in vitro assays used to determine whether a specific antibody or antigen-binding fragment has been administered include in vitro cell culture experiments in which patient tissue samples are cultured and exposed to or otherwise administered the compound, and the effect of such compound on the tissue sample is observed.

Various delivery systems are known and can be used to administer the antibodies of the present invention or polynucleotides encoding the antibodies of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.

Diagnostic methods

Overexpression of TIGIT has been observed in certain tumor samples, and patients with TIGIT-overexpressing cells may respond to treatment using the anti-TIGIT antibody of the present invention. Therefore, the antibody of the present invention can also be used for diagnosis and prognosis.

Preferably, the sample containing cells can be obtained from a patient, who may be a cancer patient or a patient awaiting diagnosis. The cells are cells from tumor tissue or tumor masses, blood samples, urine samples, or any sample from the patient. After selectively pretreating the sample, it can be incubated with the antibody of the present invention under conditions that allow the antibody to interact with the TIGIT protein that may be present in the sample. The presence of the TIGIT protein in the sample can be detected using methods such as ELISA, utilizing the anti-TIGIT antibody.

The presence (at any level or concentration) of TIGIT protein in a sample can be used to diagnose cancer, indicating whether a patient is suitable for antibody therapy or whether a patient has (or has not) responded to cancer treatment. For prognostic methods, testing can be performed once, twice, or more at specific stages at the start of cancer treatment to indicate treatment progress.

Composition

The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

In one specific implementation, the term "pharmaceutically acceptable" refers to materials listed in the pharmacopoeia for use in animals, and particularly in humans. Furthermore, "pharmaceutically acceptable carriers" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used in treatment. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline, glucose, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable drug 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, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, and tonic agents such as sodium chloride or dextran are also foreseeable. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in E.W. Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of antibody, preferably in purified form, along with a suitable number of carriers to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. Parent formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed before administration.

The compounds of the present invention can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

In the following embodiments, unless otherwise specified, the reagents and instruments used are conventional reagents and instruments in the art and can be obtained commercially; the methods used are all conventional techniques in the art, and those skilled in the art can undoubtedly implement the embodiments and obtain the corresponding results based on the description of the embodiments.

Construction of some cells:

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. Using this technique, Jurkat cells, sAPC cells, and CTI-Jurkat cells overexpressing human TIGIT were constructed.

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

Construction of CTI-Jurkat cells: The full-length human TIGIT gene (SEQ ID NO.55), the full-length human CD226 gene (SEQ ID NO.56), the IL-2 response element (SEQ ID NO.57), and the luciferase reporter gene plasmid pNF _KB -TA-luc (Beyotime, D2207) were transfected into the Jurkat cell line (ATCC, Clone E6-1, TIB-152 ^™ ) to obtain the CTI-Jurkat cell line, which can stably express human TIGIT and human CD226 molecules.

Construction of Jurkat cells overexpressing human TIGIT: The full-length human TIGIT gene (SEQ ID NO.55) was transfected into the Jurkat cell line (ATCC, Clone E6-1, TIB-152 ^™ ) to obtain the Jurkat-hTIGIT 2E6 cell line that can stably express human TIGIT.

Example

Example 1 Expression and purification of PVR-Fc and TIGIT-Fc fusion protein

The extracellular amino acid residue sequence of PVR (SEQ ID NO: 1) was optimized according to human codon preference characteristics, resulting in the nucleotide sequence SEQ ID NO: 2. Enzyme restriction sites were added, and the fusion was performed with the human IgG1 constant region sequence (SEQ ID NO: 28, nucleotide sequence shown in the underlined part of SEQ ID NO: 46) via linkers (SEQ ID NO: 6 and 7), as illustrated in Figure 1. The fused sequence was inserted into the pCDNA3.1+ vector (Invitrogen, V790-20) and then transiently transfected into 293F cells. The cell supernatant after culture was purified by Protein A affinity chromatography, and the purified fusion protein was named PVR-Fc.

The extracellular amino acid residue sequence of TIGIT (SEQ ID NO: 3) was optimized according to human codon preference characteristics, resulting in the nucleotide sequence SEQ ID NO: 48. Enzyme restriction sites were added, and the fusion was performed with the human IgG1 constant region sequence (SEQ ID NO: 28, nucleotide sequence shown in the underlined part of SEQ ID NO: 46) via linkers (SEQ ID NO: 6 and 7), as illustrated in Figure 2. The fused sequence was inserted into the pCDNA3.1+ vector and then transiently transfected into 293F cells. The cell supernatant after culture was purified by Protein A affinity chromatography, and the purified fusion protein was named TIGIT-Fc.

Table 1.1: Relevant sequences for obtaining antibodies

Example 2: Production of mouse anti-human TIGIT antibody

Female Balb/c mice aged 6-8 weeks (Guangdong Provincial Center for Medical Laboratory Animal Science) were immunized with the protein fused with the extracellular region of human TIGIT and human IgG-Fc, as described in Example 1. Two to three weeks after the initial immunization, a second immunization was performed, followed by a booster immunization approximately three weeks later. Spleen lymphocytes were then extracted and fused with myeloma cells (Sp2/0 cells, Chinese Academy of Sciences Type Culture Collection, catalog number TCM18). Hybridomas were screened based on their effective binding to TIGIT-Fc. Positive hybridomas were further subcloned through limiting dilution, and their binding affinity to TIGIT-Fc and their ability to block the binding of TIGIT-Fc and PVR-Fc were reassessed. Ultimately, six monoclonal hybridomas were obtained, one of which, 10D8, exhibited superior characteristics.

According to the method mentioned in the literature (Bradbury, A. (2010). Cloning Hybridoma cDNA by RACE. Antibody Engineering. R. Kontermann and S. Dübel. Berlin, Heidelberg, Springer Berlin Heidelberg: 15-20), specific primers were synthesized, and the target gene of the hybridoma cell antibody was cloned. The hybridoma sequencing process is as follows: 1) Hybridoma cells were revived and placed in T25 cell culture flasks with RPMI 1640 (10% FBS) medium, and then passaged for 2 generations; 2) 5 million hybridoma cells were taken, and RNA was extracted using EasyPure RNAkit (Beijing TransGen Biotech Co., Ltd., catalog number: ER101); 3) Using the extracted RNA as a template, cDNA was synthesized using a reverse transcription kit (Beijing TransGen Biotech Co., Ltd., catalog number: AT321); 4) Then, using the cDNA as a template, the target band of the antibody sequence was amplified using the synthesized specific primers. To ensure no mutations occur during amplification, FastPfu (Beijing TransGen Biotech Co., Ltd., catalog number: AP221) was used; 5) The amplified heavy and light chain target fragments were cloned into blunt-ended cloning vectors (Beijing TransGen Biotech Co., Ltd., catalog number: CB111) and then plated; 6) After the clones grew, 5 heavy chain clones and 5 light chain clones were picked and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing; 7) The sequencing results were analyzed using NCBI blasttool, NCBI IGBlast Tool and other analysis programs to find the variable region sequences of the antibody heavy chain and light chain; 8) The variable region sequence of the mouse anti-human TIGIT antibody was codon optimized based on mouse codon preference, and then the sequence was synthesized. The synthesized variable region sequence was fused with the mouse IgG2a heavy chain constant region framework (SEQ ID NO: 31, SEQ ID NO: 49) and the mouse kappa light chain constant region framework (SEQ ID NO: 32, SEQ ID NO: 50), and then cloned into the pCDNA3.1+ vector. Subsequently, the heavy chain plasmid and the light chain plasmid were transiently transfected into CHO-S cells (Bibco, catalog number: A29133) at a 1:1 ratio. The resulting antibody was the recombinant mouse anti-human TIGIT antibody, named m10D8.

The sequence of the heavy chain V region of m10D8 is shown in SEQ ID NO: 20, and the sequence of the light chain V region 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), and 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).

The antibody obtained from recombinant expression was further validated by ELISA for its binding ability with human TIGIT (see Figure 3), monkey TIGIT (see Figure 4), and mouse TIGIT (see Figure 5). The positive control antibody was 10A7 antibody (hamster anti-mouse TIGIT, see SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 28, SEQ ID NO: 30). The results showed that m10D8 could bind well to human TIGIT and monkey TIGIT, but not to mouse TIGIT. The affinity of this m10D8 antibody was determined using a Biacore-SPR instrument, and its binding constant was 1.02E+06 (1/Ms), dissociation constant was 2.00E-04 (1/s), and equilibrium dissociation constant _KD was 1.97E-10 (M).

Table 2.1: Anti-TIGIT antibodies and related CDR sequences

Example 3: Mouse anti-human TIGIT antibody blocks the binding of TIGIT to its ligand PVR

Numerous studies have shown that blocking the binding of TIGIT to its ligand PVR with anti-TIGIT antibodies can restore the function of effector T cells and NK cells. Therefore, it is necessary to conduct in vitro experiments to study whether the screened mouse anti-human TIGIT can block the binding of TIGIT to its ligand PVR.

Take an appropriate amount of PVR-Fc protein from a -80℃ freezer and biotinylate it using the Thermo Scientific Biotinylation Kit (HSulfo-NHS-LC-Biotinylation Kit, catalog number: 21435) according to the instructions. The labeled protein is named PVR-Fc-bio. The experimental procedure is as follows: 1) Coat TIGIT-Fc (2 μg/ml) with 1×PBS (phosphate buffer) overnight at 4°C; 2) Block with 3% BSA in a 37°C incubator for 2 h; 3) Dilute PVR-Fc-bio with PBS to a final concentration of 2 μg/ml, using this solution as the antibody dilution buffer; 4) Dilute m10D8 and positive control antibody 10A7 (hamster anti-mouse TIGIT, see SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 28, SEQ ID NO: 30), and negative irrelevant control antibody dilution buffer to an initial concentration of 8 μg/ml, dilute in half, add 100 μl to each well, incubate in a 37°C incubator for 2 h, and wash 8 times with PBST (PBS containing 0.05% Tween 20); 5) Add enzyme-labeled secondary antibody (Jackson Immuno Research). Inc., Catalog No.: 016-030-084 (1:10000 dilution), add 100 μl to each well and incubate at 37°C for 1 h, then wash 8 times with PBST; 6) Develop color with TMB (3,3′,5,5′-tetramethylbenzidine) chromogenic solution, 100 μl per well; 7) Stop color development with 2 MH ₂ SO ₄ and read the value at 450 nm using a microplate reader. The results are shown in Figure 6. Under the same conditions, the competitive inhibition curve of m10D8 is below that of the positive antibody 10A7, indicating that m10D8 has a stronger ability to block the binding of TIGIT to its ligand PVR compared to the positive antibody.

Example 4: Detection of the biological function of mouse anti-human TIGIT antibody

Simulated complete activation of naïve T cells requires the joint participation of both a first signal and a second signal. Based on this principle, a method for detecting the biological activity of anti-TIGIT antibodies was developed. This detection system uses two cell types: artificially constructed antigen-presenting cells (sAPCs) overexpressing mouse anti-human CD3 single-chain antibody and PVR; and engineered Jurkat cells (CTI-Jurkat) overexpressing TIGIT and CD226, which are also transfected with a luciferase reporter gene detection element that relies on the IL-2 promoter for transcription-translation initiation. The first signal is the binding of the anti-human CD3 single-chain antibody on the sAPC cell membrane to the T cell receptor (TCR) on the Jurkat cell membrane, activating T cells. The second signal is the signal transduction triggered by the interaction between PVR on the sAPC cell membrane and the activating receptor CD226 or the repressor receptor TIGIT on Jurkat cells. In the absence of anti-TIGIT antibodies, PVR preferentially binds to TIGIT because its affinity for TIGIT is much higher than that for CD226. This triggers TIGIT intracellular domain signaling, inhibiting T cell proliferation and cytokine release. Simultaneously, PVR blocks the formation of functional dimers of CD226 on the same cell surface, affecting the normal physiological function of CD226. Furthermore, because CD226 has a weaker affinity for PVR, it cannot compete with TIGIT under the same conditions, thus failing to effectively activate T cells. As a result, T cells are generally in an inhibited state, and the IL-2 promoter cannot effectively initiate the transcription and translation of the luciferase reporter gene. Conversely, when anti-TIGIT antibodies are present in the detection system, the high-affinity anti-TIGIT antibodies can bind to TIGIT, thereby preventing the signal transduction between TIGIT and PVR. At the same time, TIGIT cannot block CD226 dimerization, so CD226 can effectively bind to PVR, thereby enabling the intracellular domain signal of CD226 to be transduced and activate T cells. The IL-2 promoter effectively initiates the transcription and translation of the luciferase reporter gene, and with the participation of the luciferase substrate, it can spontaneously emit a fluorescent signal that can be captured by the instrument.

Prepare sAPC cells. Culture sAPCs to the logarithmic growth phase in CD CHO AGT ^™ medium (containing 25 μM MSX and 400 μg/ml hygromycin), then centrifuge at 800 rpm for 5 minutes. Resuspend sAPCs in Ham's F-12 medium (containing 10% FBS) to 400,000/ml. Then, use a multipipe to seed 100 μl of the medium into the corresponding wells of a 96-well plate, i.e., 40,000 cells per well. Incubate overnight in an 8% _CO2 incubator at 37°C. The next day, discard the medium and invert the cell culture plate onto absorbent paper to blot away any remaining medium.

Prepare CTI-Jurkat cells. Culture CTI-Jurkat cells to the logarithmic growth phase in 1640 medium containing 10% FBS. Then, resuspend the CTI-Jurkat cells in 1640 (1% FBS) medium to 5 million/ml. Finally, use a multi-channel pipette to add 40 μl of CTI-Jurkat cells to the wells of an sAPC, i.e., 200,000 cells per well.

Prepare the antibodies. Dilute m10D8, the positive control antibody Tiragolumab (see SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 30), and the negative control antibody BAT5906 (see Chinese patent application CN110003328A) with 1640 (1% FBS) medium to set three concentrations of 64, 32, 16, and 0 μg/ml. Then add 40 μl of the diluted antibody to each well containing sAPC and CTI-Jurkat cells (i.e., final antibody concentrations of 32, 16, 8, and 0 μg/ml).

After incubating the cell culture plates with the added samples in an 8% _CO2 incubator at 37°C for 4 hours, they were removed and allowed to return to room temperature for 10 minutes. Then, 80 μl of luciferase substrate (taken from a -20°C freezer 2 hours prior and allowed to reach room temperature) was added to each well. The culture plates were placed in a microplate reader, shaken for 5 seconds, and the fluorescence values were read after 10 minutes (see Figure 7). The results showed that both Tiragolumab and m10D8 could activate T cells, but under the same conditions, m10D8 exhibited the strongest T cell activation ability.

Example 5: Humanization of mouse anti-human TIGIT antibody

The heavy chain and light chain variable regions of the mouse anti-human TIGIT antibody were compared with sequences in the human antibody database, and the results are shown in Table 5.1. The humanization process must balance drug-likeness and potential immunogenicity while ensuring high antibody affinity. Based on bioinformatics modeling calculations, three mutants were designed for the first humanization of the m10D8 humanized antibody: h10D8V1 (see SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 29, SEQ ID NO: 30), h10D8V2 (see SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 28, SEQ ID NO: 30), and h10D8V3 (see SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 28, SEQ ID NO: 30). The light chain variable region framework of h10D8V1 and h10D8V2 is IGKV4-1*01, and the heavy chain variable region framework is IGHV3-23*01. The heavy chain variable region framework of h10D8V3 is IGHV3-64*04, and the light chain variable region framework is IGKV1-NL1*01. Binding experiments were performed on the mutant proteins with TIGIT, and biological activity was also assessed. The purified h10D8V1, h10D8V2, and h10D8V3 were used in binding experiments with TIGIT. ELISA results (see Figure 8) showed that the EC50 (concentrations eliciting a 50% maximal effect) for the three antibodies were 0.07623 μg/ml, 0.09241 μg/ml, and 0.05259 μg/ml, respectively. Based on EC50, h10D8V3 appears to have a stronger binding affinity to TIGIT. However, ELISA results reflect the static antigen-antibody binding and do not dynamically reflect the binding and dissociation of antibodies and antigens. Therefore, the equilibrium dissociation constants (KD) of these three antibodies with the TIGIT-histag antigen were determined using SPR-Biacore technology (see Figure 9 and Table 5.2). These results show that h10D8V3 has a slower binding rate and a faster dissociation rate compared to the other two antibodies, resulting in a higher equilibrium dissociation constant value, indicating lower affinity. The variable regions of h10D8V1 and h10D8V2 are completely identical. The only difference is a two-amino acid residue difference at positions 356 (Eu-encoded, corresponding to position 377 in Kabat) and 358 (Eu-encoded, corresponding to position 381 in Kabat) of the constant region. Therefore, the difference in affinity is not significant. Furthermore, the biological activities of h10D8V1, h10D8V2, and h10D8V3 were compared using methods described in Example 4. The results (see Figure 10) show that h10D8V1, h10D8V2, and h10D8V3 have similar abilities to activate T cells, indicating that their biological activities are similar.

Table 5.1 Comparison results of murine antibodies with the human germline gene database (IMGT Scientific Chart)

Table 5.2 Determination of equilibrium dissociation constants of h10D8V1, h10D8V2, h10D8V3, and TIGIT-his tag antigen

Based on the results of h10D8V1, h10D8V2, and h10D8V3, a second humanization design was performed on m10D8, resulting in three more mutants: h10D8OF (see 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 (see SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 28, SEQ ID NO: 30), and h10D8V5 (see SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 28, SEQ ID NO: 30). The heavy chain framework of h10D8OF is IGHV3-64*04, and the light chain framework is IGKV3D-7*01. The heavy chain framework of h10D8V4 is IGHV3-64*04, and the light chain framework is IGKV4-1*01. Its main feature is that some amino acid residues in the framework are consistent with those of the murine antibody m10D8. The heavy chain and light chain frameworks of h10D8V5 are the same as those of h10D8V4. Its main feature is that the interface between the antibody and the antigen is as consistent as possible with the interface between the murine antibody m10D8 and TIGIT. The binding capabilities of h10D8OF, h10D8V4, and h10D8V5 to TIGIT were compared laterally, as shown in Figure 11. The results showed that the binding curves of h10D8OF, h10D8V4, and h10D8V5 basically overlapped, and the EC50 values were 0.02329 μg/ml, 0.02560 μg/ml, and 0.02408 μg/ml, respectively.

The binding abilities of h10D8OF and h10D8V2 to TIGIT were compared. The binding curves of the two showed a high degree of overlap. The EC50 value of h10D8OF was slightly lower than that of h10D8V2, suggesting that h10D8OF might have a slightly higher affinity based solely on the ELISA results (see Figure 12). The EC50 values of h10D8OF, h10D8V2, and Tiragolumab were 0.04273 μg/ml, 0.05310 μg/ml, and 0.05361 μg/ml, respectively. The equilibrium dissociation constants K _{_D} of h10D8OF, h10D8V2, and Tiragolumab with the TIGIT-his tag antigen were also detected using SPR-Biacore technology (see Figure 13 and Table 5.3). Dynamic affinity testing revealed that h10D8V2 has a higher affinity for monovalent TIGIT-his than h10D8OF. The biological activities of h10D8OF, h10D8V4, and h10D8V5 were also tested using methods similar to those in Example 4. The results (see Figure 14) showed that at a higher concentration (32 μg/ml), h10D8OF had a stronger ability to restore T cell function, while the results for h10D8V4 and h10D8V5 were very similar.

Table 5.3 Determination of equilibrium dissociation constants of h10D8OF, h10D8V2, and TIGIT-his tag antigen (a)

(a) Note: The TIGIT-his tag used in this table is from a different batch of antigen than that used in Table 5.2, so the results are somewhat different.

Table 5.4: Humanized Sequences

Example 6: Humanized Antibody Blocking of TIGIT and PVR Binding Experiment

Theoretically, humanized murine antibodies retain some of their functions, such as blocking the binding of TIGIT to PVR. The experimental method for blocking TIGIT-PVR binding with humanized antibodies is similar to that in Example 3. The ability of h10D8OF, h10D8V4, h10D8V5, Tiragolumab (positive control antibody), and the isotype control BAT5906 to block TIGIT-PVR binding under the same conditions was tested by ELISA. The experimental results (see Figure 15) show that h10D8OF, h10D8V4, h10D8V5, and Tiragolumab can all effectively block the binding of TIGIT to PVR, with IC50 values (concentrations resulting in 50% inhibition) of 0.1592 μg/ml, 0.2166 μg/ml, 0.2837 μg/ml, and 0.4289 μg/ml, respectively. Based on the IC50 value and the upper and lower plateau values of the 4-parameter curve, h10D8OF and h10D8V4 have a stronger ability to block the binding of TIGIT to PVR than the positive antibody Tiragolumab.

Furthermore, the ability of these antibodies to block the binding of biotinylated PVR to TIGIT on the surface of Jurkat cells was further verified using Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT 2E6). The reaction system was as follows: The antibody was diluted with 80 nM biotinylated PVR-Fc protein solution prepared with PBS. The initial antibody concentration was 15 nM, and it was halved to set 10 concentration gradients (including 0 nM). The 10 concentration gradients of antibody were aliquoted into 10 centrifuge tubes, each containing 1 million Jurkat-hTIGIT cells. The cells were then resuspended and incubated at 4°C for 1 hour. After centrifugation, the supernatant of each tube was discarded, and the cells were washed twice with PBS. Then, 100 μl of Streptavidin PE (Thermo, catalog number: 12-4317-87) diluted 1:2000 was added to each tube, and the cells were resuspended and incubated at 4°C for 40 minutes. After centrifugation, the supernatant was discarded from each tube of cells. The cells were washed twice with PBS and resuspended in 200 μl of PBS. The fluorescence value (mean FL2-A) was then detected using flow cytometry. Finally, the collected fluorescence values were analyzed using a 4-parameter fitting plot to calculate the IC50 value. The IC50 values of h10D8OF, h10D8V4, and h10D8V5 (see Figure 16) were 11.26 nM, 15.78 nM, and 10.44 nM, respectively. All three antibodies effectively blocked the binding of biotinylated PVR-FC to TIGIT on the Jurkat-hTIGIT cell membrane, with h10D8OF and h10D8V5 showing better blocking effects. The ability of h10D8OF to block the binding of PVR to TIGIT was also compared with that of the positive antibody Tiragolumab (see Figure 17), with h10D8OF showing a stronger blocking effect.

Example 7: Detection of humanized antibody ADCC activity

Jurkat cells (ATCC, Clone E6-1, catalog number: TIB-152 ^™ ) were transfected with NFAT, luciferase reporter gene elements, and the FcγRIIIa (CD16) receptor gene. This successfully constructed stable cell line stably expresses the FcγRIIIa receptor, an essential molecule mediating ADCC biological activity. By connecting effector cells to target cells with a monoclonal antibody, the NFAT pathway is activated, initiating luciferase reporter gene expression. Jurkat cells (ATCC, Clone E6-1) were transfected with the human TIGIT gene to overexpress the human TIGIT molecule; this stable cell line served as the target cell line. The procedure for detecting antibody ADCC activity was as follows: 1) An appropriate amount of cells was taken according to the cell density, centrifuged at 1200 rpm for 5 min to remove the growth medium, and then the cell density was adjusted. 2) The two cell types were mixed and plated into a cell culture plate (Corning, catalog number: 3917). Effector cells: 80,000 per well; target cells: 20,000 per well; total volume of both types of cells per well: 50 μl. 3) Dilute the antibody using 1% FBS 1640 as the dilution medium. For h10D8OF, Tiragolumab, and irrelevant antibody (without ADCC activity), start at a concentration of 1.8 μg/ml (50 μl per well, final concentration 0.9 μg/ml), dilute 1/3, with 3 replicates per concentration gradient, and the last gradient at 0 μg/ml. 4) After adding the antibody, gently tap the plate to mix the sample. Then incubate at 37℃ in an 8% _CO2 incubator for 4 hours. 5) After incubation, allow to stand at room temperature for 10 minutes. The luciferase substrate reagent should also be equilibrated to room temperature beforehand. Add 100 μl of substrate reaction solution to each well, and start reading the values after 10 minutes. The results are shown in Figure 18. Both h10D8OF and Tiragolumab have ADCC activity, and under the same conditions, h10D8OF has slightly better ADCC activity than Tiragolumab.

In another experiment, the host cells CHO that secrete h10D8OF were modified (modification methods are described in patents CN107881160A and WO2019029713 A1) to remove fucose from the h10D8OF antibody, and the antibody was named h10D8OFKF. The difference in ADCC activity between h10D8OFKF and h10D8OF was compared using a similar method. The results are shown in Figure 19. Both h10D8OFKF and h10D8OF exhibited ADCC activity, with EC50 values of 0.0152 nM and 0.125 nM, respectively. Under the same conditions, the ADCC activity of h10D8OFKF was 8.8 times that of h10D8OF, suggesting that stronger ADCC activity may result in better in vivo efficacy.

Example 8: Cytokine Release Experiment under the Action of Humanized Anti-TIGIT Antibody

TIGIT, upon binding to PVR (CD155) expressed on the surface of tumor cells or APC cells, downregulates T cell effector function and suppresses anti-tumor immune responses. The ability of anti-TIGIT antibodies to block PVR-mediated inhibition of T cell cytokine release was investigated. Purchased primary human CD3+ cells (Guangzhou Rede Biotechnology Co., Ltd., catalog number: 1507) were induced and cultured for 10 days in 10% FBS 1640 medium containing PHA (2 μg/ml) and IL-2 (4 ng/ml). The cells were then centrifuged and resuspended in PHA- and IL-2-free medium, and then placed in a 5% CO2 incubator at 37°C for 24 hours. Cells were then resuspended at 5 × 10^5 cells/ml and incubated at room temperature for 30 min with the irrelevant antibody BAT5906 (see Chinese patent application CN110003328A), h10D8OF (25 μg/ml and 10 μg/ml), and Tiragolumab (25 μg/ml and 10 μg/ml). After incubation, cells were seeded into cell culture plates pre-coated with anti-CD3 antibody (10 μg/ml) (BD Biosciences, catalog number 555336), PVR-Fc (10 μg/ml), or a combination of anti-CD3 antibody (10 μg/ml) and PVR-Fc (10 μg/ml), and then incubated in a cell culture incubator (37°C, 5% _CO2 ) for 48 hours. Finally, cells were removed from the cell culture plates, centrifuged, and the supernatant was retained. The supernatant was diluted several times and the release of cytokines IFN-γ and IL-2 in the cell supernatant was detected.

Using the cytokines produced by lymphocytes induced by anti-CD3 antibody alone as a baseline, the ratios of cytokines induced by other treatments were calculated, with the former set at 1.0 and the latter expressed as actual ratios. The results are shown in Figures 20 and 21. The results indicate that h10D8OF induces lymphocytes to produce more IFN-γ and IL-2 with increasing concentration. Example 9 validated the tumor-suppressive ability of the anti-TIGIT antibody using a B-hPD-1/hTIGIT dual-humanized mouse model inoculated with MC38 tumor cells.

Since humanized antibodies do not recognize mouse TIGIT, the extracellular regions of PD-1 and TIGIT molecules in the genome of C57BL/6 background mice were humanized to replace human PD-1 and human TIGIT, resulting in B-hPD-1/hTIGIT dual-humanized mice. The mouse strain is C57BL/6-Pdcd1tm1(hPDCD1)Tigit tm1(hTIGIT)/Bcgen (Biocytogene Biotechnology Co., Ltd.). In the spleen cells of B-hPD-1/hTIGIT homozygous mice, hTIGIT and hPD-1 mRNA expression were detected, but mTIGIT and mPD-1 mRNA expression was not detected (data from the Biocytogene Biotechnology Co., Ltd. website). An MC38 colon cancer animal model was established using B-hPD-1/hTIGIT humanized mice to verify the efficacy of anti-TIGIT antibodies and the efficacy of combined anti-TIGIT and anti-PD-1 antibody therapy.

Before conducting animal pharmacodynamic experiments, preliminary experiments were performed to verify that the anti-TIGIT antibody could effectively bind to hTIGIT on the cell surface of B-hPD-1/hTIGIT dual-humanized mice. 11.5 μg of anti-mCD3e antibody (Biolegend, catalog number: 100301) was dissolved in 300 μl of PBS. A B-hPD-1/hTIGIT dual-humanized mouse was then intraperitoneally injected with 200 μl of the anti-mCD3e antibody solution. 24 h later, the mouse was euthanized by cervical dislocation, and the spleen was quickly removed and placed in a 15 ml centrifuge tube containing 5 ml of PBS. The spleen was then transferred to a 6-well plate and placed on a 70 μm sieve. 1 ml of PBS was added, and the cells were ground using the end of a sterile syringe. The sieve was then rinsed with 1-2 ml of PBS. The filtered cell suspension was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and then briefly centrifuged again to remove the supernatant. Then, 1 ml of erythrocyte lysis buffer (Beyotime, catalog number: C3702) was added to each spleen, and the cells were lysed on ice for 5 min. Finally, 5 ml of PBS was added, the cells were mixed, centrifuged at 3000 rpm at 4°C for 3 min, the supernatant was discarded, and then the supernatant was briefly removed again. Next, flow cytometry analysis was performed. The cells were first suspended in 300 μl of PBS, and 6 μl of anti-mCD16/32 antibody (Biolegend, #101310) and Human TruStrain FcX (Biolegend, catalog number: 422302) were added, mixed, and incubated on ice for 15 min. Add 1.2 ml of PBS and mix well to adjust the cell concentration to 4 × 10^7/ml. Add 25 μl of cell suspension to the tubes to be stained. Add the corresponding antibodies PerCP-anti-mTcRβ (Biolegend, catalog number: 109228) and APC-anti-hTIGIT antibody APC-anti-hTIGIT (eBioscience, catalog number: 17-9500-42) to each tube, along with mouse anti-TIGIT antibody secondary antibody Anti-mouse IgG, Alexa F... Fluor 647 is Goat Anti-Mouse IgG/Alexa Fluor 647 (Beijing Bio-Sens Biotechnology Co., Ltd., catalog number: bs-0296G-AF647), a humanized anti-TIGIT antibody secondary antibody (anti-human Fc). FITC is Goat anti-Human IgG Fc secondary antibody, treated with FITC (Invitrogen, catalog number A18818) before detection. The experimental design is shown in Table 9.1. Flow cytometry results (see Figure 22) show that m10D8, h10D8V1, h10D8OF, and Tiragolumab can all effectively bind to hTIGIT on the surface of B-hPD-1/hTIGIT dual-humanized mouse T cells. Under the same conditions, h10D8OF has a stronger binding ability to hTIGIT than other antibodies. Similar results were observed in the binding assays of h10D8OF, Tiragolumab, and Jurkat cells overexpressing human TIGIT to Jurkat-hTIGIT 2E6 (see Figure 23), which may indicate that h10D8OF (EC50 value 0.6961 nM) has a stronger affinity for TIGIT in physiological or near-physiological states compared to Tiragolumab (EC50 value 1.906 nM).

Mice, aged 6-8 weeks, were all female, with n=6 mice per group. MC38 cells (5× ^10⁵ cells/0.1ml/mice) were subcutaneously injected into the right axilla of each mouse. Treatment began when the tumor volume reached 150±50 mm³. Mice with tumors that were too large or too small were culled. Treatment was administered every 3 days, and tumor volume was measured twice weekly. The dosage of Pembrolizumab was 0.1 mg/kg, and the dosages of Tiragolumab and m10D8 were both 20 mg/kg. The efficacy results of anti-TIGIT antibody monotherapy (see Figure 24) showed that both Tiragolumab and m10D8 antibodies could inhibit tumor growth, and the efficacy of m10D8 monotherapy was similar to that of Tiragolumab. The efficacy results of humanized anti-TIGIT antibody monotherapy (see Figure 25) show that h10D8V2 has a poor tumor-inhibiting effect, while h10D8OF has a similar tumor-inhibiting effect to tiragolumab. The efficacy results of combined anti-TIGIT antibody and pembrolizumab (see Figure 26) show that the combined use of h10D8V2 and pembrolizumab has a poor tumor-inhibiting effect, while the combined use of h10D8OF and pembrolizumab has a similar efficacy to tiragolumab and pembrolizumab. Overall, from an animal efficacy perspective, both murine and humanized antibodies have tumor-inhibiting effects and show potential for combined use with pembrolizumab.

Table 9.1 Experimental study on the binding of anti-TIGIT antibody to hTIGIT on the surface of B-hPD-1/hTIGIT dual-humanized mouse T cells.

a) The reagent antibody (eBioscience, catalog number: 7-9500-42)

Example 10: Effects of humanized anti-TIGIT antibody on lymphocyte subsets

At the treatment endpoint of the in vivo pharmacodynamic study, the effects of anti-TIGIT antibody on CD4+ T cells and CD8+ T cells in mouse MC38 tumor-infiltrating lymphocytes (TILs) were analyzed. Tumors were collected from mice in the Vehicle control group (injection saline) (n=4) and the h10D8OF (20 mg/kg) monotherapy group (n=5). Fresh tumor tissue was surgically removed from the mice, washed with PBS, and stored on ice. The tissue was then repeatedly cut with ophthalmic scissors and poured into an IKA grinder. It was ground at 1200 rpm for 10 seconds each, for a total of 2 minutes. The ground tumor tissue solution was filtered through a 70 μm sieve into a 50 mL centrifuge tube, and the tissue was washed once more. The tube was then centrifuged at 4°C, 2000 rpm for 5 minutes, the supernatant was discarded, and the tumor cells were resuspended in an appropriate amount of PBS.

First, dead/live cell staining was performed. 2 μl of Zombie NIR ^™ dye (Biolegend #423106) was added to 500 μl of PBS to prepare a 2× dead/live cell staining solution. In a 96-well plate, 25 μl of tumor cell suspension was added to each well of the experimental group. 25 μl of the 2× dead/live cell staining solution was then added to the corresponding well containing the tumor cell suspension. The plates were then gently incubated at room temperature in the dark for 15 min. Next, cell surface staining was performed. 25 μl of the stained cells from the previous step were transferred to a new 96-well plate using a pipette. 25 μl of cell surface labeling molecule staining solution was added to each well. The plates were incubated at 4°C in the dark for 30 min. Cell surface marker staining solutions 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 incubation, cells were resuspended in 185 μl of PBS, mixed, and centrifuged at 2000 rpm for 5 min at 4°C. The supernatant was discarded, and the cells were gently tapped onto absorbent paper to remove any remaining liquid. Then, 200 μl of fixative (1×) was added to each well to resuspend the cells, mixed, and incubated overnight at 4°C. After fixation, cells were centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the cells were gently tapped onto absorbent paper to remove any remaining liquid. Add 200 μl of transmembrane transfer solution to each well, centrifuge at 3000 rpm for 5 min, discard the supernatant, gently tap the plate on absorbent paper, and discard any residual liquid. Resuspend the cells in 25 μl of transmembrane transfer solution to each well, and simultaneously add 25 μl of prepared intracellular staining solution (Anti-Mouse/Rat Foxp3, APC), incubate at 4°C in the dark for 30 min. Add 200 μl of 1× transmembrane transfer solution to each well, mix well, centrifuge at 3000 rpm for 5 min at 4°C, discard the supernatant, gently tap the plate on absorbent paper, and discard any residual liquid. Resuspend the cells in 250 μl of PBS, and analyze according to the Attune NXT flow cytometer SOP.

First, mCD45+ leukocytes were sorted from the prepared single cells (see Figure 27). Then, CD4+ T cells (see Figure 28) and CD8+ T cells (see Figure 29) within the mCD45+ cells were analyzed. The results showed no significant difference in mCD45+ lymphocytes between the two groups of mouse TILs. Further analysis revealed no significant difference in CD4+ T cells and CD8+ T cells either. Next, further analysis of TIGIT+CD4+ T cells and TIGIT+CD8+ T cells (see Figures 30 and 31) showed that, compared to the control group, the proportions of TIGIT+CD4+ T cells and TIGIT+CD8+ T cells were significantly decreased in the single-drug treatment group. This result indicates that under the action of anti-TIGIT antibody, the expression of TIGIT molecules in CD4+ T cells and CD8+ T cells of mouse MC38 colon cancer tumor TILs is likely downregulated, and it also indirectly indicates that the function of CD8+ T cells was restored.

Example 11 validates the tumor-suppressive ability of the anti-TIGIT humanized antibody using B-hTIGIT humanized mice and hPD-L1 MC38 tumor models.

Since the anti-TIGIT humanized antibody does not recognize mouse TIGIT, the extracellular region of the TIGIT molecule in the genome of C57BL/6 background mice was humanized to replace human TIGIT, resulting in B-hTIGIT humanized mice. The mouse strain was C57BL/6-Tigit ^tm1(hTIGIT) /Bcgen (Biocytogen Biotechnology Co., Ltd.). hTIGIT mRNA expression was detected in the spleen cells of B-hTIGIT homozygous mice, but mTIGIT mRNA expression was not detected (data from the Biocytogen Biotechnology Co., Ltd. website). This experiment used B-hTIGIT humanized mice and an MC38 tumor model overexpressing hPD-L1 to verify the tumor-suppressive ability of the anti-TIGIT humanized antibody and the potential synergistic effect of combined administration of anti-TIGIT and anti-PD-L1 antibodies.

To enable h10D8OF and Tiragolumab to exert the most effective ADCC function in mice, the heavy chain constant region of h10D8OF and Tiragolumab was replaced with the heavy chain constant region of mouse antibody IgG2a (SEQ ID NO: 31), and the light chain constant region of h10D8OF and Tiragolumab was replaced with the light chain constant region of mouse antibody IgG2a (SEQ ID NO: 32). The recombinant antibodies were named mh10D8OF and mTiragolumab.

Six- to eight-week-old B-hTIGIT humanized mice were selected. MC38-hPD-L1 cells (MC38 cells expressing human PD-L1 while simultaneously knocking out mouse PD-L1) resuspended in PBS (Biocytogene Biotechnology Co., Ltd.) were subcutaneously injected into the right side of each B-hTIGIT humanized mouse at a concentration of 5 × ^10⁵ cells/0.1 mL, with a volume of 0.1 mL per mouse. When the average tumor volume reached 100-150 ^mm³ , mice with suitable tumor volume and body weight were selected and randomly assigned to six experimental groups, with ten mice in each group. The administration route was intraperitoneal injection. The control group consisted of Vehicle (physiological saline for injection), and two groups of mh10D8OF were administered at doses of 10 mg/kg and 30 mg/kg, respectively. The mTiragolumab dose was 30 mg/kg, and the anti-PD-L1 antibody Atezolizumab dose was 1 mg/kg. The combined administration group received mh10D8OF at 30 mg/kg and anti-PD-L1 antibody Atezolizumab at 1 mg/kg. The experimental results are shown in Figure 32.

It can be seen that both mh10D8OF and mTiragolumab effectively inhibited tumor growth, with no statistically significant difference in the degree of inhibition; both had a TGI of over 50%. TGI (Tumor Growth Inhibition value) is the tumor (volume) inhibition rate. It is used to evaluate the inhibitory effect of a drug on tumor growth in vivo (i.e., in 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 dosage settings, no significant synergistic effect was observed in this experiment. However, from a biological perspective, the combined administration at this dosage condition resulted in a more significant inhibition of tumor growth, with a tumor inhibition rate (TGI) of 66%.

Claims (69)

1. An antibody or a fragment thereof, characterized in that the antibody or fragment thereof specifically binds to an immunoglobulin and an immunoreceptor tyrosine inhibitory motif of a T cell immune receptor (TIGIT) protein, wherein the antibody or fragment thereof comprises VH CDR1 as shown in SEQ ID NO: 21, VH CDR2 as shown in SEQ ID NO: 22, VH CDR3 as shown in SEQ ID NO: 23, VL CDR1 as shown in SEQ ID NO: 25, VL CDR2 as shown in SEQ ID NO: 26 or 43, and VL CDR3 as shown in SEQ ID NO: 27. 2. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof is an isotype of IgG, IgM, IgA, IgE or IgD. 3. The antibody or fragment thereof as claimed in claim 1, wherein the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. 4. The antibody or fragment thereof as described in claim 3, wherein the Fc region is the human Fc region. 5. The antibody or fragment thereof as described in claim 4, characterized in that, according to the Eu numbering system, the amino acid at position 356 of the human Fc region heavy chain is E. 6. The antibody or fragment thereof as described in claim 4, characterized in that, according to the Eu numbering system, the amino acid at position 386 of the human Fc region heavy chain is M. 7. The antibody or fragment thereof as claimed in claim 4, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or 29. 8. The antibody or fragment thereof as claimed in claim 4, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO: 30. 9. The antibody or fragment thereof as described in any one of claims 1 to 8, wherein the antibody or fragment thereof is a humanized antibody. 10. The antibody or fragment thereof as claimed in claim 1, characterized in that 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 NO: 20, 33, 35, 37, 39 and/or 41, or a peptide having at least 90% sequence homology with an amino acid sequence consisting of the group consisting of SEQ ID NO: 20, 33, 35, 37, 39 and/or 41. 11. The antibody or fragment thereof as claimed in claim 1, characterized in that 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 NO: 24, 34, 36, 38, 40 and/or 42, or a peptide having at least 90% homology with an amino acid sequence consisting of the group consisting of SEQ ID NO: 24, 34, 36, 38, 40 and/or 42. 12. An antibody or a fragment thereof, characterized in that the antibody or fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 35 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 36. 13. The antibody or fragment thereof according to any one of claims 10-12, characterized in that the heavy chain portion 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. 14. The antibody or fragment thereof according to any one of claims 10-12, characterized in that the heavy chain portion 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. 15. An antibody or a fragment thereof, characterized in that the antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:44, and the antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO:45. 16. The antibody or fragment thereof as described in any one of claims 1 to 8, 10 to 12, and 15, characterized in that the antibody or fragment thereof can effectively block the binding of TIGIT to PVR. 17. The antibody or fragment thereof as claimed in claim 9, wherein the antibody or fragment thereof can effectively block the binding of TIGIT to PVR. 18. The antibody or fragment thereof as claimed in claim 13, wherein the antibody or fragment thereof can effectively block the binding of TIGIT to PVR. 19. The antibody or fragment thereof as claimed in claim 14, wherein the antibody or fragment thereof can effectively block the binding of TIGIT to PVR. 20. The antibody or fragment thereof as described in any one of claims 1 to 8, 10 to 12, and 15, characterized in that the antibody or fragment thereof can activate lymphocytes to release cytokines. 21. The antibody or fragment thereof as claimed in claim 9, wherein the antibody or fragment thereof can activate lymphocytes to release cytokines. 22. The antibody or fragment thereof as claimed in claim 13, wherein the antibody or fragment thereof can activate lymphocytes to release cytokines. 23. The antibody or fragment thereof as claimed in claim 14, wherein the antibody or fragment thereof can activate lymphocytes to release cytokines. 24. The antibody or fragment thereof as described in any one of claims 1 to 8, 10 to 12, and 15, characterized in that the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 100 pM. 25. The antibody or fragment thereof as claimed in claim 9, wherein the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 100 pM. 26. The antibody or fragment thereof as claimed in claim 13, wherein the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 100 pM. 27. The antibody or fragment thereof as claimed in claim 14, wherein the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 100 pM. 28. The antibody or fragment thereof as described in any one of claims 1 to 8, 10 to 12, and 15, characterized in that the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 5 nM. 29. The antibody or fragment thereof as claimed in claim 9, wherein the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 5 nM. 30. The antibody or fragment thereof as claimed in claim 13, wherein the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 5 nM. 31. The antibody or fragment thereof as claimed in claim 14, characterized in that the affinity of the antibody or fragment thereof to human TIGIT is _KD ≤ 5 nM. 32. The antibody or fragment thereof as described in any one of claims 1 to 8, 10 to 12, 15, 17 to 19, 21 to 23, 25 to 27, 29 to 31, characterized in that the antibody or fragment thereof has ADCC activity. 33. The antibody or fragment thereof as claimed in claim 9, wherein the antibody or fragment thereof has ADCC activity. 34. The antibody or fragment thereof as claimed in claim 13, wherein the antibody or fragment thereof has ADCC activity. 35. The antibody or fragment thereof as claimed in claim 14, wherein the antibody or fragment thereof has ADCC activity. 36. The antibody or fragment thereof as claimed in claim 16, wherein the antibody or fragment thereof has ADCC activity. 37. The antibody or fragment thereof as claimed in claim 20, wherein the antibody or fragment thereof has ADCC activity. 38. The antibody or fragment thereof as claimed in claim 24, wherein the antibody or fragment thereof has ADCC activity. 39. The antibody or fragment thereof as claimed in claim 28, wherein the antibody or fragment thereof has ADCC activity. 40. The antibody or fragment thereof as described in any one of claims 1 to 8, 10 to 12, 15, 17 to 19, 21 to 23, 25 to 27, 29 to 31, 33 to 39, characterized in that the antibody or fragment thereof is not bound to fucose. 41. The antibody or fragment thereof as claimed in claim 9, wherein the antibody or fragment thereof is not bound to fucose. 42. The antibody or fragment thereof as claimed in claim 13, wherein the antibody or fragment thereof is not bound to fucose. 43. The antibody or fragment thereof as claimed in claim 14, wherein the antibody or fragment thereof is not bound to fucose. 44. The antibody or fragment thereof as claimed in claim 16, wherein the antibody or fragment thereof is not bound to fucose. 45. The antibody or fragment thereof as claimed in claim 20, wherein the antibody or fragment thereof is not bound to fucose. 46. The antibody or fragment thereof as claimed in claim 24, wherein the antibody or fragment thereof is not bound to fucose. 47. The antibody or fragment thereof as claimed in claim 28, wherein the antibody or fragment thereof is not bound to fucose. 48. The antibody or fragment thereof as claimed in claim 32, wherein the antibody or fragment thereof is not bound to fucose. 49. A bispecific antibody, characterized in that the bispecific antibody comprises an antibody fragment as described in any one of claims 1 to 48 and a second antigen-binding fragment that is specific to molecules on immune cells. 50. The bispecific antibody of claim 49, 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. 51. A composition, characterized in that the composition comprises an antibody or a fragment thereof as described in any one of claims 1 to 48 or a bispecific antibody as described in claim 49 or 50, and a pharmaceutically acceptable carrier. 52. A cell, characterized in that the cell comprises one or more polynucleotides encoding an antibody or a fragment thereof as described in any one of claims 1 to 48 or a bispecific antibody as described in claim 49 or 50. 53. Use of the antibody or fragment thereof according to any one of claims 1 to 48, or the bispecific antibody according to claim 49 or 50, in the preparation of a medicament for treating cancer or infection. 54. The use as described in claim 53, wherein the cancer is a solid tumor. 55. The use as described in claim 53, wherein the cancer is selected from bladder cancer, liver 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, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. 56. The use as described in claim 55, wherein the gastrointestinal cancer is colon cancer, rectal cancer, or gastric cancer. 57. The use as described in any one of claims 53 to 56, characterized in that the treatment further comprises administering a second cancer treatment agent to the patient. 58. The use as described in claim 53, wherein the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. 59. Use of the antibody or fragment thereof according to any one of claims 1-48, or the bispecific antibody according to claim 49 or 50, in the preparation of a medicament for treating cancer or infection, wherein the treatment comprises: (a) In vitro, cells are treated with the said antibody or a fragment thereof, or the said bispecific antibody, and (b) The treated cells were then administered to the patient. 60. The use as claimed in claim 59, characterized in that the treatment further includes isolating the cells from the individual prior to step (a). 61. The use as described in claim 60, wherein the cells are isolated from the patient. 62. The use as claimed in claim 60, wherein the cells are isolated from a donor individual different from the patient. 63. The use as described in any one of claims 59 to 62, wherein the cell is a T cell. 64. The use as described in claim 63, wherein the T cell is a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof. 65. Use of the antibody or fragment thereof of any one of claims 1 to 48, or the bispecific antibody of claim 49 or 50, in the preparation of a product for detecting TIGIT expression in a sample, said product being used in a method comprising: contacting a sample with the antibody or fragment thereof of any one of claims 1 to 48, or the bispecific antibody of claim 49 or 50, such that the antibody or fragment thereof or the bispecific antibody binds to TIGIT, and detecting the binding reflecting the level of TIGIT expression in the sample. 66. The use as described in claim 65, wherein the sample comprises tumor cells, tumor tissue, infected tissue, or blood sample. 67. A polynucleotide, characterized in that the polynucleotide can encode an antibody or a fragment thereof as described in any one of claims 1 to 48 or a bispecific antibody as described in claim 49 or 50. 68. A method for preparing an antibody or fragment thereof according to any one of claims 1 to 48 or a bispecific antibody according to claim 49 or 50, characterized in that cells comprising a polynucleotide encoding the antibody or fragment thereof or the bispecific antibody are cultured to express the antibody or fragment thereof or the bispecific antibody. 69. The method according to claim 68, wherein the cells are CHO cells, HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, or mouse L cells.