Anti-TIGIT antibodies, their preparation and application

Abstract

An isolated antigen-binding protein is provided, the antigen-binding protein comprising at least one CDR in a heavy chain variable region VH, the VH comprising the amino acid sequence set forth in SEQ ID NO: 55. Also provided are anti-TIGIT antibodies, methods for preparing the same, and applications thereof. The antigen-binding protein can specifically bind to the TIGIT antigen, inhibit the binding of TIGIT to its ligand, and can be used to prepare drugs for preventing or treating TIGIT-related diseases.

Description

[Technical field]

[0001] The present invention relates to the field of biopharmaceuticals, specifically to an antigen-binding protein targeting TIGIT, its preparation method and use. [Background technology]

[0002] Normally, T cells react to exogenous or endogenous antigen-MHC-I / II complexes presented by antigen-presenting cells (APCs) with T cells (CD4 + , CD8 + ) T cells bind to a surface receptor (T cell receptor, TCR) and, after being fully activated through additional costimulatory signals, kill cells expressing related antigens by other effector cells. Conversely, T cells are also regulated by coinhibitory signals, the latter of which have a negative regulatory effect on T cell activity, proliferation, survival, etc., and can maintain immune tolerance, prevent excessive autoimmune reactions, and reduce tissue damage caused by immune responses. In the tumor cell microenvironment, T cell activation and inhibition are in an abnormal pattern, and its normalization is now widely recognized as an important means of tumor treatment.

[0003] TIGIT (also known as T cell Ig and ITIM domain, WUCAM, Vstm3, and VSIG9) is a member of the poliovirus receptor (PVR) / Nectin family. It consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with classical immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoglobulin tyrosine tail (ITT) motifs. TIGIT is expressed on lymphocytes and specifically binds to effector and regulatory CD4 + T cells, follicular helper CD4 + T cells, effector CD8 +It is highly expressed in T cells and natural killer (NK) cells. The TIGIT gene is located on human chromosome 16 and encodes a type I transmembrane protein of 244 amino acids. The extracellular domain of the human TIGIT molecule is 141 amino acids long and contains one immunoglobulin V-like domain. The transmembrane region contains 23 amino acids. The cytoplasmic domain is short, containing 80 amino acids, and contains one PDZ-binding domain and one ITIM motif.

[0004] CD155 (also called PVR, Necl5 or Tage4) is a high affinity ligand for TIGIT. CD155, which is highly expressed on the tumor surface, binds to TIGIT on the surface of NK and T cells, suppressing their killing action against tumor cells. TIGIT can inhibit the effect of NK cells by preventing the early death of tumor cells and the release of tumor antigens. It inhibits the costimulatory ability of dendritic cells, resulting in a decrease in cancer antigen presentation and an increase in anti-inflammatory cytokines such as IL-10. TIGIT can also induce PVR signaling in other cells, such as tumor cells. TIGIT is a CD8 + They can directly block the effects of T cells or TIGIT + Tregs are CD8 + It can also inhibit T cells and prevent the elimination of cancer cells (Manieri et al., Inhibition of Cancer Immunity Cycle by TIGIT. 2016).

[0005] Numerous preclinical results suggest that TIGIT may be a suitable target for patients with cancer and other diseases; therefore, given the role and function of TIGIT in various related diseases, there remains a strong need in the field for the development of improved anti-TIGIT-specific antibodies suitable for patient treatment. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0006] The present application provides an isolated antigen binding protein that may comprise at least one CDR in a heavy chain variable region VH, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:55. The isolated antigen binding protein may comprise at least one CDR in a light chain variable region VL, wherein the VL may comprise the amino acid sequence shown in SEQ ID NO:64. The isolated antigen binding protein has one or more of the following properties: 1) a 1×10 -10 M or less (e.g., 8×10 -11 M, 5×10 -11 M, 2×10 -11 M, 1×10 -11 M or less), which is determined by surface plasmon resonance; 2) it can block the binding of TIGIT to CD155 in FACS analysis; and 3) it can inhibit tumor growth and / or tumor cell proliferation. [Means for solving the problem]

[0007] On the other hand, the present application provides an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH, said VH may comprise the amino acid sequence shown in SEQ ID NO:55.

[0008] In some embodiments, the isolated antigen binding protein comprises at least one CDR in a light chain variable region, VL, wherein the VL may comprise the amino acid sequence set forth in SEQ ID NO:64.

[0009] In some embodiments, the isolated antigen binding protein has one or more of the following properties: 1) a concentration of 1×10 -10 1) the ability to bind to TIGIT protein with a KD value of M or less, wherein the KD value is determined by surface plasmon resonance; 2) the ability to block binding of CD155 to TIGIT in a FACS assay; and 3) the ability to inhibit tumor growth and / or tumor cell proliferation.

[0010] In some embodiments, the isolated antigen binding protein competes with a reference antibody for binding to the TIGIT protein, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:56, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:57, and the light chain variable region of the reference antibody may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:65, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:66.

[0011] In some embodiments, the isolated antigen binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0012] In some embodiments, the antigen-binding fragment may comprise a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab.)2, a scFv, a di-scFv, and / or a dAb.

[0013] In some embodiments, the antibody is selected from the group consisting of: a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0014] In some embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3.

[0015] In some embodiments, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:56.

[0016] In some embodiments, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NOs:4 and 42.

[0017] In some embodiments, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57.

[0018] In some embodiments, the HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs:5 and 43.

[0019] In some embodiments, the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, and the isolated antigen binding protein is selected from any set of amino acid sequences below:

[0020] (1) HCDR1: SEQ ID NO: 3, HCDR2: SEQ ID NO: 4, and HCDR3: SEQ ID NO: 5, and (2) HCDR1: SEQ ID NO:3, HCDR2: SEQ ID NO:42, and HCDR3: SEQ ID NO:43.

[0021] In some embodiments, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:65.

[0022] In some embodiments, the LCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:6 and 52.

[0023] In some embodiments, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7.

[0024] In some embodiments, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66.

[0025] In some embodiments, the LCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:8 and 53.

[0026] In some embodiments, the isolated antigen binding protein comprises an LCDR1, an LCDR2 and an LCDR3, and the isolated antigen binding protein is selected from any set of amino acid sequences below.

[0027] (1) LCDR1:SEQ ID NO:6, LCDR2:SEQ ID NO:7, and LCDR3:SEQ ID NO:8, and (2) LCDR1: SEQ ID NO: 52, LCDR2: SEQ ID NO: 7, and LCDR3: SEQ ID NO: 53. In some embodiments, the VL may include framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0028] In some embodiments, the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and said L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:68.

[0029] In some embodiments, the L-FR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 15, 16, 44, and 45.

[0030] In some embodiments, the L-FR2 is located between LCDR1 and LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:70.

[0031] In some embodiments, the L-FR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 18, 46, and 47.

[0032] In some embodiments, said L-FR3 is located between LCDR2 and LCDR3, and said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:72.

[0033] In some embodiments, the L-FR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 19, 20, 21, 48, 49, and 50.

[0034] In some embodiments, the N-terminus of said L-FR4 is linked to the C-terminus of LCDR3, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:73.

[0035] In some embodiments, the L-FR4 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:22 and 51.

[0036] In some embodiments, the VL may comprise the amino acid sequence shown in SEQ ID NO:64.

[0037] In some embodiments, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 2, 10, 11, 13, 33, and 34.

[0038] In some embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, wherein the antibody light chain constant region is derived from a human κ light chain constant region, and wherein the human κ light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31.

[0039] In some embodiments, the VH may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0040] In some embodiments, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:58.

[0041] In some embodiments, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:23 and 35.

[0042] In some embodiments, the H-FR2 is located between HCDR1 and HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:60.

[0043] In some embodiments, the H-FR2 is located between HCDR1 and HCDR2, and the H-FR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:24, 25, 26, 36, and 37.

[0044] In some embodiments, the H-FR3 is located between HCDR2 and HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:61.

[0045] In some embodiments, the H-FR3 is located between HCDR2 and HCDR3, and the H-FR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 27, 38, and 39.

[0046] In some embodiments, the N-terminus of said H-FR4 is linked to the C-terminus of HCDR3, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:62.

[0047] In some embodiments, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 28, 40, and 41.

[0048] In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:55.

[0049] In some embodiments, the VH may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1, 9, 12, 14, and 32.

[0050] In some embodiments, said isolated antigen binding protein comprises an antibody heavy chain constant region, and said antibody constant region is derived from a human IgG constant region.

[0051] In some embodiments, the antibody heavy chain constant region comprises a human IgG1 constant region, and the human IgG1 constant region may comprise an amino acid sequence set forth in any one of SEQ ID NOs:29-30.

[0052] In some embodiments, the antigen binding protein comprises a heavy chain variable region, VH, and a light chain variable region, VL, and the isolated antigen binding protein is selected from any of the following sets of amino acid sequences: (1) VH:SEQ ID NO:9, VL:SEQ ID NO:10, (2) VH:SEQ ID NO:9, VL:SEQ ID NO:11, (3) VH: SEQ ID NO:12, VL:SEQ ID NO:13, (4) VH:SEQ ID NO:14, VL:SEQ ID NO:11, (5) VH:SEQ ID NO:1, VL:SEQ ID NO:2, (6) VH:SEQ ID NO:32, VL:SEQ ID NO:34, and (7) VH:SEQ ID NO:32, VL: SEQ ID NO:33.

[0053] On the other hand, the present application provides an isolated nucleic acid molecule or molecules encoding said isolated antigen binding proteins.

[0054] On the other hand, the present application provides a vector, which may comprise the nucleic acid molecule.

[0055] On the other hand, the present application provides a cell, which may comprise the nucleic acid molecule or the vector.

[0056] Alternatively, the present application provides a polypeptide, which may comprise the isolated antigen-binding protein.

[0057] On the other hand, the present application provides an immunoconjugate, which may comprise said isolated antigen-binding protein or said polypeptide.

[0058] On the other hand, the present application provides a method of preparing said isolated antigen binding protein, said method may comprise culturing said cell under conditions allowing expression of said isolated antigen binding protein.

[0059] On the other hand, the present application provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein, the nucleic acid molecule, the vector, the polypeptide, the immunoconjugate and / or the cell, and optionally a pharma- ceutical acceptable adjuvant.

[0060] Meanwhile, the present application provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the polypeptide, the immunoconjugate and / or the pharmaceutical composition in the preparation of a medicament, wherein the medicament is used for the prevention, alleviation and / or treatment of a TIGIT-associated disease.

[0061] In some embodiments, the TIGIT-associated disease is a T cell dysfunction disease.

[0062] In some embodiments, the TIGIT-related disease is a tumor. In some embodiments, the tumor may include a solid tumor and / or a non-solid tumor.

[0063] In some embodiments, the tumor is selected from the group consisting of: breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, renal cancer, melanoma, T lymphoma, and pancreatic cancer.

[0064] On the other hand, the present application provides a method for inhibiting binding of CD155 to TIGIT, which may comprise administering the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the polypeptide, the immunoconjugate and / or the pharmaceutical composition.

[0065] On the other hand, the present application provides the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the polypeptide, the immune complex and / or the pharmaceutical composition for preventing, alleviating or treating TIGIT-related diseases.

[0066] On the other hand, the present application provides a method for preventing, alleviating or treating a TIGIT-associated disease, which may comprise administering to a subject in need thereof the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell, the polypeptide, the immunoconjugate and / or the pharmaceutical composition.

[0067] In some embodiments, the TIGIT-associated disease is a T cell dysfunction disease.

[0068] In some embodiments, the TIGIT-related disease is a tumor. In some embodiments, the tumor may include a solid tumor and / or a non-solid tumor.

[0069] In some embodiments, the tumor is selected from the group consisting of: breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, renal cancer, melanoma, T lymphoma, and pancreatic cancer. Effect of the Invention

[0070] Those skilled in the art can easily ascertain other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Therefore, the accompanying drawings and the description of the present application are merely illustrative and not restrictive. [Brief description of the drawings]

[0071] Specific features of the invention according to the present application are set forth in the appended claims. A better understanding of the features and advantages of the invention according to the present application can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is as follows:

[0072] [Figure 1] 1 shows the binding activity of TIGIT chimeric antibodies to cells expressing human TIGIT. [Diagram 2] 1 shows the binding activity of TIGIT humanized antibodies to cells expressing human TIGIT. [Diagram 3] 1 shows the blocking activity of a TIGIT chimeric antibody against the binding of human TIGIT to its ligand CD155. [Figure 4] 1 shows the blocking activity of TIGIT humanized antibodies against the binding of human TIGIT to its ligand CD155. [Diagram 5] FIG. 1 shows an in vivo efficacy test of TIGIT antibody in animals. [Figure 6] FIG. 1 is a diagram comparing the efficacy tests in animals of TIGIT antibody and control antibody Tiragolumab. [Figure 7] 1 shows the results of efficacy tests of TIGIT antibodies in animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] In the following, the embodiments of the present application will be described using certain specific embodiments, but those familiar with this technology can easily understand other advantages and effects of the present application from the disclosure in this application.

[0074] Term definition

[0075] In the present application, the term "TIGIT" refers to "Ig and ITIM domain-containing T cell immunoreceptor" and generally means that immunoglobulins that are members of the PVR (poliovirus receptor) family bind to PVR / CD155 and Nectin-2 / CD112. In the present application, TIGIT can refer to TIGIT protein from any vertebrate, including mammals such as primates (e.g., humans, rhesus monkeys, and cynomolgus monkeys) and rodents (e.g., mice and rats). The term includes full-length TIGIT or fragments thereof (such as mature fragments lacking a signal peptide), unprocessed TIGIT, any form of TIGIT processed in cells, and synthetic TIGIT. It also includes variants of TIGIT, such as splice variants and allelic variants. In a specific embodiment, TIGIT is human TIGIT, and the amino acid sequence of human TIGIT can include the amino acid sequence set forth in UniProt Accession No. Q495A1. In certain embodiments, TIGIT is cynomolgus monkey TIGIT, and the amino acid sequence of cynomolgus monkey TIGIT may comprise the amino acid sequence set forth in UniProt Accession No. G7NXM4. In certain embodiments, TIGIT is mouse TIGIT, and the amino acid sequence of mouse TIGIT may comprise the amino acid sequence set forth in UniProt Accession No. P86176.

[0076] In this application, the term "antigen-binding protein" generally refers to a moiety that binds to an antigen and, optionally, a scaffold or framework moiety that allows the antigen-binding protein to adopt a conformation that promotes binding of the antigen to the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, so long as they exhibit the desired antigen-binding activity.

[0077] In this application, the term "Fab" generally refers to a fragment that includes a heavy chain variable structural domain and a light chain variable structural domain, and further includes a constant structural domain of the light chain and the first constant structural domain (CH1) of the heavy chain, and the term "Fab'" generally refers to a fragment that differs from Fab by the addition of a small number of residues (including one or more cysteines from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 structural domain. The term "F(ab')2" generally refers to an antibody fragment that includes a dimer of Fab, two Fab fragments linked by disulfide bridges at the hinge region. The term "Fv" generally refers to the minimum antibody fragment that includes a complete antigen recognition and binding site. In certain embodiments, it may consist of a dimer of a heavy chain variable region and a light chain variable region in tight non-covalent association. The term "dsFv" generally refers to a disulfide-stabilized Fv fragment in which a single light chain variable region and a single heavy chain variable region are linked by disulfide bonds. The term "dAb fragment" generally refers to an antibody fragment that consists of a VH structural domain. In this application, the term "scFv" generally refers to a monovalent molecule formed by the covalent linkage of an antibody heavy chain variable domain and a light chain variable domain via a flexible peptide linker, where the scFv molecule may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker VL-COOH.

[0078] In this application, the term "antibody" is used in the broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies consisting of two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelliaized mono-structural domain antibodies. The term "antibody" may generally include a protein having at least two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain has a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region has three structural domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain may include a light chain variable region (VL) and a light chain constant region. In the light chain constant region, there is a structural domain, CL. The VH and VL regions are further subdivided into hypervariable regions called complementarity determining regions (CDRs), which alternate with more conservative regions called framework regions (FRs). Each VH and VL has three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Natural heavy and light chain variable domains each may contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), and are mostly β-folded, with the three CDRs forming loop connections and sometimes forming part of the β-fold structure. The CDRs of each chain are held in close proximity to each other through the FR regions, and together with the CDRs of the other chain, form the antigen-binding site of the antibody. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues and factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0079] In this application, the term "variable" generally refers to the fact that a portion of the sequence of the variable structural domain of an antibody is highly variable, which results in the binding and specificity of various specific antibodies to their particular antigens. However, the variability is not uniformly distributed throughout the variable region of an antibody. It is concentrated in three segments of the light and heavy chain variable regions, known as complementarity determining regions (CDRs) or highly variable regions (HVRs). The more conserved parts of the variable regions are called frame regions (FRs). In the art, the CDRs of an antibody are defined in various ways, such as the Kabat definition rules based on sequence variation (see Kabat et al., Protein Sequences in Immunology, 5th ed., National Institutes of Health, Bethesda, Maryland (1991)), the Chothia definition rules based on the position of the structural loop regions (see A1- Lazikani et al., JMol Biol 273:927-48,1997), and the IMGT scientific chart rules and IMGT definition rules based on the concepts of IMGT-ONTOLOGY.

[0080] IMGT stands for International ImMunoGeneTics Information System, a worldwide reference database for immunogenetics and immunoinformatics (http: / / www.imgt.org). IMGT specializes in human and other vertebrate immunoglobulins (IGs), antibodies, T cell receptors (TRs), major histocompatibility (MH), and vertebrate and invertebrate immunoglobulin superfamily (Igsf), MH superfamily (MhSf), and immune system related proteins (RPIs).

[0081] As used herein, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and / or recovered from components of the environment in which it is produced (e.g., naturally or recombinantly). Contaminant components of the environment in which they occur are usually substances that would interfere with research, diagnostic or therapeutic uses, and may include enzymes, hormones, other proteinaceous or non-proteinaceous solutes. Isolated antigen binding proteins or antibodies are usually prepared by at least one purification step.

[0082] As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the cluster are identical, except for a small number of natural mutations that may exist. Monoclonal antibodies are usually highly specific for a single antigenic site. Furthermore, whereas traditional polyclonal antibody preparations usually have different antibodies against different determinants, each monoclonal antibody is directed against a single cluster of determinants on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" is intended to indicate the character of the antibody obtained from a substantially homogeneous population of antibodies and does not require any particular method of production of the antibody. For example, monoclonal antibodies as used herein may be prepared in hybridoma cells or by recombinant DNA techniques.

[0083] As used herein, the term "chimeric antibody" generally refers to an antibody whose variable regions are derived from one species and whose constant regions are derived from another species. Typically, the variable regions are derived from an antibody of a laboratory animal such as a rodent (the "parent antibody"), and the constant regions are derived from a human antibody, making the resulting chimeric antibody less likely to provoke a harmful immune response in a human individual than the parent (e.g., mouse-derived) antibody.

[0084] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR domains of a non-human antibody (e.g., a murine antibody) have been replaced with the corresponding amino acids from a human immunoglobulin. Also, small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may be tolerated as long as the antibody still retains the ability to bind a particular antigen. A humanized antibody may optionally include at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains the same antigen specificity as the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies may consist minimally of chimeric antibodies derived from the sequence of a non-human immunoglobulin. In certain embodiments, residues in the CDR domains of a human immunoglobulin (acceptor antibody) may be replaced with residues in the CDR domains of a non-human species (donor antibody) (e.g., mouse, rat, rabbit or non-human primate) having the desired properties, affinities and / or capacities. In certain embodiments, residues in the FR domains of a human immunoglobulin may be replaced with the corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0085] In this application, a "fully human antibody" generally refers to an antibody expressed in an animal by introducing an antibody-encoding gene from a human into an antibody gene-deficient animal through genetic engineering. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. This can significantly reduce immune side effects. Possible methods for obtaining fully human antibodies in this field include the phage display deep Ls method, the transgenic mouse method, the ribosome display deep Ls method, and the RNA-polypeptide method.

[0086] In this application, the terms "binding", "specific binding" or "specific for" generally refer to a measurable and reproducible interaction, e.g., the binding of an antigen to an antibody, which allows the presence of a target to be determined in the presence of a heterologous molecule (including a biomolecule). For example, an antibody binds to an epitope via its antigen-binding domain, which requires some complementarity between the antigen-binding domain and the epitope. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to this target with higher affinity, affinity, more readily, and / or for a longer period of time than it binds to other targets. An antibody is said to "specifically bind" to an antigen when it binds to the epitope via its antigen-binding domain more readily than to unrelated epitopes that it randomly binds. An "epitope" is a specific group of atoms (such as a sugar side chain, a phosphate group, a sulfonyl group, etc.) or amino acid on an antigen that binds to an antigen-binding protein (such as an antibody).

[0087] In this application, "K D The terms " and "KD" are used interchangeably and generally refer to the equilibrium dissociation constant, where "KD" refers to the ratio of the dissociation rate constant (kdis, also known as "off-rate (koff)" or "kd") to the association rate constant (kon, also known as "on-rate (kon)" or "ka"). The binding affinity of an antigen-binding protein (such as an antibody) to an antigen can be expressed using the association rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD). Methods for determining association and dissociation rate constants are well known in the art and include, but are not limited to, biofilm interferometry (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technologies. The affinity of a particular protein-protein interaction measured may differ if measured under different conditions (e.g., salinity, pH).

[0088] In this application, the term "reference antibody" generally refers to an antibody with which an antigen-binding protein described in this application competes for binding to the antigen TIGIT.

[0089] As used herein, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" generally refers to a form of cytotoxicity in which numerous secreted immunoglobulins bind to Fc receptors (FcR) on certain cytotoxic effector cells (e.g., NK cells, neutrophils, and macrophages) and these cytotoxic effector cells can specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxic agents. The primary cells mediating ADCC (e.g., NK cells) express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII (see Table 3 on page 464 of Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991)). To assess the ADCC activity of a target molecule, in vitro and / or in vivo cytotoxicity assays can be performed, for example, in vitro ADCC assays can be performed as described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or 6,737,056 (Presta). Effector cells that can be used in such assays include PBMCs and NK cells. Alternatively / additionally, the ADCC activity of a target molecule can be assessed in vivo, for example, in an animal model as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) and retains FcRn binding ability.

[0090] ADCC activity can be reduced by modifying the Fc region. In certain embodiments, sites that affect binding to Fc receptors can be removed, e.g., sites that are not salvage receptor binding sites. In certain embodiments, the Fc region can be modified to remove ADCC sites. ADCC sites are known in the art, see, e.g., Sarmay et al. (1992) Molec. Immunol. 29 (5):633-9 for the ADCC site of IgG1.

[0091] In the present application, "between..." generally refers to the C-terminus of an amino acid fragment being directly or indirectly linked to the N-terminus of a first amino acid fragment, and the N-terminus being directly or indirectly linked to the C-terminus of a second amino acid fragment. In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2. For example, the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3. In the heavy chain, for example, the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2. For example, the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR3. In the present application, the "first amino acid fragment" and the "second amino acid fragment" may be either the same or different amino acid fragments.

[0092] As used herein, the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and / or recovered from components of the environment in which it is produced (e.g., naturally or recombinantly). Contaminant components of the environment in which they occur are usually substances that would interfere with research, diagnostic or therapeutic uses and may include enzymes, hormones, other proteinaceous or non-proteinaceous solutes. Isolated antigen binding proteins or antibodies are usually prepared by at least one purification step. Isolated antigen binding proteins as described herein generally do not bind to antigens that are not TIGIT antigens.

[0093] As used herein, the term "isolated nucleic acid molecule" or "isolated polynucleotide" refers to DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or any combination thereof, that is not linked to all or part of a polynucleotide found in nature.

[0094] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host cell and transferring an inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is primarily used to insert DNA or RNA into a cell, a vector that is primarily used to replicate DNA or RNA, and a vector that is primarily used for transcriptional and / or translational expression of DNA or RNA. The vector also includes vectors having the various functions described above. The vector may be a polynucleotide that is transcribed or translated into a polypeptide when introduced into a suitable host cell. Usually, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0095] As used herein, the term "cell" generally refers to an individual cell, cell line, or cell culture that can contain or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or that can express an antibody or antigen-binding fragment thereof described herein. The cell may be comprised of the progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the daughter cells may not necessarily be morphologically or genomically identical to the original parent cell, but it is sufficient that they are capable of expressing an antibody or antigen-binding fragment thereof described herein. The cell may include a cell transfected in vitro with a vector described herein. The cell may be a prokaryotic cell (e.g., E. coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell, or a myeloma cell). In certain embodiments, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell. As used herein, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cells include not only the particular cells but also the progeny of those cells.

[0096] In this application, the term "pharmaceutical acceptable adjuvant" may include pharmaceutical acceptable vectors, excipients or stabilizers that are generally non-toxic to exposed cells or mammals at the doses and concentrations used. Typically, physiologically acceptable vectors are pH-buffered aqueous solutions. Examples of physiologically acceptable vectors include buffers such as phosphate, citrate, and other organic acids, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) peptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, carbohydrates such as monosaccharides and disaccharides such as glucose, mannose, and dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol and sorbitol, salt-forming counterions such as sodium, and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0097] As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic, diagnostic, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. The terms "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with the cell, contact of a reagent with a fluid, and contact of a fluid with the cell. The terms "administration" and "treatment" may also refer to by binding a reagent, diagnostic, composition, or other in vitro and isolated treatment of cells. "Treatment," as applied to a human, animal, or research subject, refers to therapeutic treatment, prophylactic treatment, research, and diagnosis, and includes contact of a TIGIT conjugate with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0098] In the present application, the term "treatment" refers to the administration of a therapeutic agent, including any of the TIGIT antigen binding proteins and compositions thereof, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, either orally or topically. The patient is typically administered an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount). The therapeutic effect may be a reduction in the rate of disease progression, an improvement or alleviation of the disease state, remission or improved prognosis, etc. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated. This includes, but is not limited to, a reduction in the proliferation (or destruction) of cancer cells, a reduction in symptoms resulting from the disease, an improvement in the quality of life of an individual with the disease, a reduction in the dosage of other drugs required to treat the disease, a delay in the progression of the disease, and / or an extension of the individual's survival.

[0099] As used herein, the term "tumor" refers to all redundant (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0100] As used herein, the terms "optionally" or "optionally" refer to the subsequently described event or circumstance that may occur, but does not necessarily occur.

[0101] In this application, the term "comprise" generally means to include, include, contain, or cover. In some cases, it can also mean "for" or "consisting of."

[0102] As used herein, the term "about" generally refers to a variation within a 0.5%-10% range above or below a specified numerical value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified nominal value.

[0103] Detailed Description of the Invention

[0104] Antigen-binding proteins

[0105] On the other hand, the present application provides an isolated antigen binding protein which may comprise at least one CDR in a heavy chain variable region VH, said VH may comprise the amino acid sequence shown in SEQ ID NO:55.

[0106] On the other hand, the present application provides an isolated antigen binding protein which may comprise at least one CDR in a heavy chain variable region VH, said VH may comprise the amino acid sequence shown in SEQ ID NO:54.

[0107] In the present application, the isolated antigen binding protein may comprise at least one CDR in a heavy chain variable region VH, wherein the VH may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 9, 12, 14 and 32.

[0108] In the present application, the VH of the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3.

[0109] The antigen binding protein described herein may comprise an HCDR1, which may comprise the amino acid sequence shown in SEQ ID NO:3: GYSITSDYA, e.g., a sequence determined according to the IMGT definition rules.

[0110] The antigen binding protein described herein may comprise an HCDR2, which may comprise the amino acid sequence shown in SEQ ID NO:56: IX2X3SGX6X7, where X2 may be S or T, X3 may be S or Y, X6 may be A or S, and X7 may be P or T. For example, the sequence may be determined according to the IMGT definition rules.

[0111] For example, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs:4 and 42.

[0112] The antigen binding protein described herein may comprise an HCDR2, which may comprise the amino acid sequence shown in SEQ ID NO:4: ITSSGST, e.g., a sequence determined according to the IMGT definition rules.

[0113] The antigen binding protein described herein may comprise an HCDR3, wherein said HCDR3 has the amino acid sequence shown in SEQ ID NO:57: AX2LX4X5X6X7YX9X 10 X2 is R or S, X4 is D or G, X5 is F or T, X6 is D or G, X7 is N or Y, X9 is G or absent, and X 10 is G or absent. For example, it may be a sequence determined according to the IMGT definition rules.

[0114] For example, the HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs:5 and 43.

[0115] The antigen binding protein described herein may comprise an HCDR3, which may comprise the amino acid sequence shown in SEQ ID NO:5: ARLDFGNYGGAMDY, e.g., a sequence determined according to the IMGT definition rules.

[0116] The antigen binding proteins described herein may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.

[0117] The antigen binding protein described herein may comprise a framework region H-FR1, said H-FR1 having the amino acid sequence shown in SEQ ID NO:58: X1VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 where X1 is D or Q, X 16 is E, Q, or X 17 is S or T, and X 25may be S or T. For example, it may be a sequence determined according to the IMGT definition rules.

[0118] For example, the H-FR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:23 and 35.

[0119] The antigen binding protein described herein may comprise a frame region H-FR1, said H-FR1 comprising the amino acid sequence shown in SEQ ID NO:23: QVQLQESGPGLVKPSETLSLTCTVS, e.g., a sequence determined according to the IMGT definition rules.

[0120] The antigen binding protein described herein may comprise a framework region H-FR2, said H-FR2 having the amino acid sequence shown in SEQ ID NO:60: WX2WIRQX7PGX 10 X 11 X 12 E.W.X. 15 wherein X2 is I or N, X7 is F or P, X 10 is K, N, or X 11 is G, R or K, X 12 is L, V, X 15 may be I or M. For example, it may be a sequence determined according to the IMGT definition rules.

[0121] For example, the H-FR2 may comprise an amino acid sequence shown in any one of SEQ ID NOs: 24-26 and 36-37.

[0122] The antigen binding protein described herein may comprise a framework region H-FR2, said H-FR2 having the amino acid sequence shown in SEQ ID NO:59: WIWIRQPPGX 10 X 11 LEWIGY, where X 10 is K, N, or X 11 may be G or K. For example, it may be a sequence determined according to the IMGT definition rules.

[0123] For example, the H-FR2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs:2426.

[0124] The antigen binding protein described herein may comprise a framework region H-FR3, said H-FR3 having the amino acid sequence shown in SEQ ID NO:61: X1YNPSLKSRX 10 X 11 X 12 X 13 X 14 DTSKNQFX 22 LX 24 LX 26 X 27 VTX 30 X 31 DTATYYC, where X1 is R, S or Y, 10 is I or V, X 11 is S, T, X 12 is F, I, X 13 is S, T, X 14 is R, V, X 22 is F, S, X 24 is K, Q, or X 26 is S, T, X 27 is F, S, X 30 is A or T, and X 31 may be A or E. For example, it may be a sequence determined according to the IMGT definition rules.

[0125] For example, the H-FR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 27, 38, and 39.

[0126] The antigen binding protein described herein may comprise a framework region H-FR3, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:27: YNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC, e.g., a sequence determined according to the IMGT definition rules.

[0127] The antigen binding protein described herein may comprise a framework region H-FR4, said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:62: WGQGTX6VX8VSS, where X6 may be L or S and X8 may be I or T. For example, the sequence may be determined according to the IMGT definition rules.

[0128] For example, the H-FR4 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:28, 40, and 41.

[0129] The antigen binding protein described herein may comprise a framework region H-FR4, said H-FR4 comprising the amino acid sequence shown in SEQ ID NO:28: WGQGTLVTVSS, e.g., a sequence determined according to the IMGT definition rules.

[0130] The antigen binding protein described herein may comprise a light chain variable region VH, said VH having the amino acid sequence shown in SEQ ID NO:55: X1VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 GYSITSDYAWX 36 WIRQX 41 PGX 44 X 45 X 46 E.W.X. 49 GYIX 53 X 54 SGX 57 X 58 X 59 YNPSLKSRX 68 X 69 X 70 X 71 X 72 DTSKNQFX 80 LX 82 LX 84 X 85 VTX 88 X 89 DTATYYCAX 98 LX100X 101 X 102 X 103 YX 105 X106 AMDYWGQGTX 116 VX 118 VSS, where X1 is D or Q, X 16 is E, Q, or X 17 is S, T, X 25 is S, T, X 36 is I, N, or X 41 is F, P, X 44 is N, K, X 45 is G, K or R, X 46 is L, V, X 49 is I, M, or X 53 is S, T, X 54 is S, Y, X 57 is A, S, or X 58 is P, T, X 59 is R, S or Y, X 68 is I or V, X 69 is S, T, X 70 is F, I, X 71 is S, T, X 72 is R, V, X 80 is F, S, X 82 is K, Q, or X 84 is S, T, X 85 is F, S, X 88 is A, T, or X 89 is A, E, or X 98 is R, S, or X 100 is D, G, or X 101 is F, T, X 102 is D, G, or X 103 is N or Y. X 105 is G or not present, X 106 is G or not present, X 116 is L or S, and X 118 may be I or T. For example, it may be a sequence determined according to the IMGT definition rules.

[0131] For example, the antigen binding protein may comprise a heavy chain variable region, VH, wherein the VH may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 1, 9, 12, 14 and 32.

[0132] The antigen binding protein described herein may comprise a light chain variable region VH, said VH having the amino acid sequence shown in SEQ ID NO:54: QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX 44 X 45 LEWIGYITSSGSTYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQTLVTVSS, where: 44 is K, N, or X 45 may be G or K. For example, it may be a sequence determined according to the IMGT definition rules.

[0133] For example, the antigen binding protein may comprise a heavy chain variable region, VH, wherein the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs:9, 12 and 14.

[0134] The antigen-binding protein described herein may comprise a heavy chain constant region, which may be derived from human IgG, e.g., human IgG1. In certain embodiments, the Fc domain of human IgG1 may be modified to achieve desired properties (e.g., ADCC KO). The modification may be an amino acid mutation. In certain embodiments, the IgG1 modification may be L234A / L235A, i.e., according to EU numbering, amino acids 234 and 235 are mutated from leucine (L) to alanine (A), respectively. In certain embodiments, the heavy chain constant region of the antigen-binding protein described herein may be derived from wild-type human IgG1.

[0135] For example, the antigen binding protein described herein may comprise a heavy chain constant region, which may comprise the amino acid sequence shown in any one of SEQ ID NOs:35-36.

[0136] In the present application, the isolated antigen binding protein may comprise at least one CDR in a light chain variable region, VL, which may comprise the amino acid sequence shown in SEQ ID NO:64.

[0137] In the present application, the isolated antigen binding protein may comprise at least one CDR in a light chain variable region, VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:63.

[0138] In the present application, the isolated antigen binding protein may comprise at least one CDR in a light chain variable region VL, wherein the VL may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 10, 11, 13, 33 and 34.

[0139] In the present application, the VL of said antigen binding protein may comprise LCDR1, LCDR2 and LCDR3.

[0140] The antigen binding protein described herein may comprise an LCDR1, said LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:65: QHVSX5A, where X5 may be N or T. For example, it may be a sequence determined according to the IMGT definition rules.

[0141] For example, the LCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NOs:6 and 52.

[0142] The antigen binding protein described herein may comprise an LCDR1, which may comprise the amino acid sequence shown in SEQ ID NO:6: QHVSTA, e.g., a sequence determined according to the IMGT definition rules.

[0143] The antigen binding protein described herein may comprise an LCDR2, said LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7: SAS, e.g., a sequence determined according to the IMGT definition rules.

[0144] The antigen binding protein described herein may comprise an LCDR3, said LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66: QQX3YX5X6PX8T, where X3 may be H or Y, X5 may be I or S, X6 may be L or T, and X8 may be W or Y. For example, the sequence may be determined according to the IMGT definition rules.

[0145] For example, the LCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs:8 and 53.

[0146] The antigen binding protein described herein may comprise an LCDR3, said LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8: QQHYITPYT, e.g., a sequence determined according to the IMGT definition rules.

[0147] The antigen binding proteins described herein may comprise the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.

[0148] The antigen binding protein described herein may comprise a framework region L-FR1, wherein said L-FR1 has the amino acid sequence shown in SEQ ID NO:68: DIX3MTQSX8X9X 10 X 11 X 12 X 13 SX 15 GDRVX 20 ITCX 24 X3 may contain Q or V, X8 may contain H or P, X9 may contain K or S, X 10 is F, or S, X 11 L, M, X 12 is F, S, X 13 is A, T, or X 15 is I or V, X 20 is S, T, X 24 may be K or R. For example, it may be a sequence determined according to the IMGT definition rules.

[0149] For example, the L-FR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 15, 16, 44, and 45.

[0150] The antigen binding protein described herein may comprise a framework region L-FR1, said L-FR1 having the amino acid sequence shown in SEQ ID NO:67: DIQMTQSPSSLSASVGDRVTITCX 24 AS, where X 24 may be K or R. For example, it may be a sequence determined according to the IMGT definition rules.

[0151] For example, the L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs:15 and 16.

[0152] The antigen binding protein described herein may comprise a framework region L-FR2, said L-FR2 having the amino acid sequence shown in SEQ ID NO:70: X1X2WYQQKPGX 10 X11PKLIX 17 where X1 is L or V, X2 is A or N, X 10 is K, Q, or X 11 is A or S, and X 17 may be H or Y. For example, it may be a sequence determined according to the IMGT definition rules.

[0153] For example, the L-FR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 18, 46, and 47.

[0154] The antigen binding protein described herein may comprise a frame region L-FR2, said L-FR2 comprising the amino acid sequence shown in SEQ ID NO:69: X1X2WYQKPGKAPKLLIY, where X1 may be L or V and X2 may be A or N. For example, the sequence may be determined according to the IMGT definition rules.

[0155] For example, the L-FR2 may comprise an amino acid sequence as shown in any one of SEQ ID NOs:17 and 18.

[0156] The antigen binding protein described herein may comprise a framework region L-FR3, said L-FR3 having the amino acid sequence shown in SEQ ID NO:72: YX2X3X4GVPX8RFX 11 GX 13 X 14 SGTDFTX 21 TIX 24 SX 26 QX 28 EDX 31 AX 33 X2 may be L or R, X3 may be Q or Y, X4 may be S or T, X8 may be D or S, 11 is I, S or T, X 13 is R, S, or X 14 is G, R, X 21 is F, L, X 24 is N, S, or X 26 is L, V, X 28 is A, P, or X 33 is F, L, X 14 may be T or V. For example, it may be a sequence determined according to the IMGT definition rules.

[0157] For example, the L-FR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 19-21 and 48-50.

[0158] The antigen binding protein described herein may comprise a framework region L-FR3, said L-FR3 having the amino acid sequence shown in SEQ ID NO:71: YX2X3SGVPSRFSGSX 14 SGTDFTLTISSLQPEDFATYYC, where X2 is L or R, X3 is Q or Y, 14 may be G or R. For example, it may be a sequence determined according to the IMGT definition rules.

[0159] For example, the L-FR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NOs:19-21.

[0160] The antigen binding protein described herein may comprise a framework region L-FR4, said L-FR4 comprising the amino acid sequence shown in SEQ ID NO:73: FGX3GTKLEIK, where X3 may be G or Q. For example, the sequence may be determined according to the IMGT definition rules.

[0161] For example, the L-FR4 may comprise an amino acid sequence as shown in any one of SEQ ID NOs:22 and 51.

[0162] The antigen binding protein described herein may comprise a framework region L-FR4, said L-FR4 comprising the amino acid sequence shown in SEQ ID NO: 22: FGQGTKLEIK, e.g., a sequence determined according to the IMGT definition rules.

[0163] The antigen binding protein described herein may comprise a light chain variable region VL, said VL having the amino acid sequence shown in SEQ ID NO:64: DIX3MTQSX8X9X 10 X 11 X 12 X13SX 15 GDRVX 20 ITCX 24 ASQHVSX 31 AX 33 X 34 WYQQKPGX 42 X 43 PKLLIX 49 SASYX 54 X 55 X 56 GVPX 60 RFX 63 GX 65 X 66 SGTDFTX 73 TIX 76 SX 78 QX 80 EDX 83 AX 85 YYCQQX91 YX 93 X 94 PX 96 TFGX 100 GTKLEIK, where X3 is Q or V, X8 is H or P, X9 is K or S, 10 is F or S, and X 11 L, M, X 12 is F, S, X 13 is A, T, or X 15 is I or V, X 20 is S, T, X 24 is K, R, X 31 is N, T, X 33 is L, V, X 34 is A, N, or X 42 is K, Q, or X 43 is A, S, or X 49 is H, Y, X 54 L or R, X 55 Q or Y, X 56 S, T, X 60 D, S, or X 63 I, S or T, X 65 S, R, X 66 G or R, X 73 L or F, X 76 S, N, X 78 L, V, X 80 A or P, X 83 F, L, X 85 T, V, X 91 H or Y, X 93 I, S, or X 94 L or TX 96 is W or Y, X 100 may be Q or G. For example, it may be a sequence determined according to the IMGT definition rules.

[0164] For example, the antigen binding protein may comprise a light chain variable region, VL, wherein the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2, 10, 11, 13, 33 and 34.

[0165] The antigen binding protein described herein may comprise a light chain variable region VL, said VL having the amino acid sequence shown in SEQ ID NO:63: DIQMTQSPSSLSASVGDRVTITCX. 24 ASQHVSTAX 33 X 34 WYQKPGKAPKLLIYSASYX 54 X 55 SGVPSRFSGSX 66 SGTDFTLTISSLQPEDFATYYCQHYITPYTFGQTKLEIK, where X 24 is K, R, X 33 is L, V, X 34 is A, N, or X 54 is L or R, X 55 is Q, Y, X 66 may be G or R. For example, it may be a sequence determined according to the IMGT definition rules.

[0166] For example, the antigen binding protein may comprise a light chain variable region, VL, wherein the VL may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10, 11 and 13.

[0167] The antigen binding protein described herein may comprise a light chain constant region, which may comprise a human light chain constant region sequence. For example, it may comprise a human kappa light chain constant region. For example, the light chain constant region of the isolated antigen binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO:31.

[0168] In the present application, said isolated antigen binding protein may comprise antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, wherein said HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, said HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:56 and said HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57.

[0169] For example, in the present application, the isolated antigen binding protein may comprise an antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, wherein said HCDR1 may comprise the amino acid sequence as set forth in SEQ ID NO:3, said HCDR2 may comprise the amino acid sequence as set forth in any one of SEQ ID NOs:4 and 42, and said HCDR3 may comprise the amino acid sequence as set forth in any one of SEQ ID NOs:5 and 43.

[0170] In the present application, said isolated antigen binding protein may comprise antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, said HCDR1, said HCDR2 and said HCDR3 may comprise the amino acid sequences as shown in SEQ ID NOs: 3, 4 and 5, respectively, in that order.

[0171] In the present application, said isolated antigen binding protein may comprise antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, said HCDR1, said HCDR2 and said HCDR3 may comprise the amino acid sequences as shown in SEQ ID NOs: 3, 42 and 43, respectively, in that order.

[0172] In the present application, the isolated antigen binding protein may comprise an antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, wherein said LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:65, said LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7 and said LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66.

[0173] For example, in the present application, the isolated antigen binding protein may comprise an antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, wherein said LCDR1 may comprise the amino acid sequence as set forth in any one of SEQ ID NOs:6 and 52, said LCDR2 may comprise the amino acid sequence as set forth in SEQ ID NO:7, and said LCDR3 may comprise the amino acid sequence as set forth in any one of SEQ ID NOs:8 and 53.

[0174] In the present application, said isolated antigen binding protein may comprise an antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, said LCDR1, said LCDR2 and said LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively, in that order.

[0175] In the present application, said isolated antigen binding protein may comprise an antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, said LCDR1, said LCDR2 and said LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs: 52, 7 and 53, respectively, in that order.

[0176] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, said HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:56, said HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57, said LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:65, said LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7, and said LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66.

[0177] For example, in the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:3, said HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs:4 and 42, said HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs:5 and 43, said LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs:6 and 52, said LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:7, and said LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs:8 and 53.

[0178] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 may comprise the amino acid sequences as set forth in SEQ ID NOs:3, 4, 5, 6, 7, 8, respectively, in that order.

[0179] In certain embodiments, the isolated antigen binding protein described herein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein said HCDR1, said HCDR2, said HCDR3, said LCDR1, said LCDR2 and said LCDR3 may comprise the amino acid sequences set forth in SEQ ID NOs: 3, 42, 43, 52, 7, 53, respectively, in that order.

[0180] In the present application, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:55, and the VL may comprise the amino acid sequence shown in SEQ ID NO:64.

[0181] In certain embodiments, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 9, 12, 32, and 14, and the VL may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 10, 11, 33, 34, and 13.

[0182] In the present application, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:54, and the VL may comprise the amino acid sequence shown in SEQ ID NO:63.

[0183] In certain embodiments, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise an amino acid sequence set forth in any one of SEQ ID NOs:9, 12, and 14, and the VL may comprise an amino acid sequence set forth in any one of SEQ ID NOs:10, 11, and 13.

[0184] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:9, and the VL may comprise the amino acid sequence shown in SEQ ID NO:10.

[0185] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:9, and the VL may comprise the amino acid sequence shown in SEQ ID NO:11.

[0186] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:12, and the VL may comprise the amino acid sequence shown in SEQ ID NO:13.

[0187] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:14, and the VL may comprise the amino acid sequence shown in SEQ ID NO:11.

[0188] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:1, and the VL may comprise the amino acid sequence shown in SEQ ID NO:2.

[0189] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:32, and the VL may comprise the amino acid sequence shown in SEQ ID NO:34.

[0190] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO:32, and the VL may comprise the amino acid sequence shown in SEQ ID NO:33.

[0191] The isolated antigen binding protein described herein may further comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region may be derived from a human IgG1 constant region and the light chain constant region may be derived from a human kappa light chain constant region.

[0192] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, where the VH may comprise the amino acid sequence set forth in SEQ ID NO:9 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:10, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain variable region, where the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:29 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as HB0030.

[0193] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, where the VH may comprise the amino acid sequence set forth in SEQ ID NO:9 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:11, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain constant region, where the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:29 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as HB0031.

[0194] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, where the VH may comprise the amino acid sequence set forth in SEQ ID NO:12 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:13, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain constant region, where the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:29 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as HB0032.

[0195] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, where the VH may comprise the amino acid sequence set forth in SEQ ID NO:14 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:11, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain constant region, where the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:29 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as HB0033.

[0196] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence set forth in SEQ ID NO:1 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:2, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:30 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as 900424.

[0197] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence set forth in SEQ ID NO:32 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:34, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:30 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as 900423.

[0198] For example, the isolated antigen binding protein may comprise a heavy chain variable region, VH, and a light chain variable region, VL, wherein the VH may comprise the amino acid sequence set forth in SEQ ID NO:32 and the VL may comprise the amino acid sequence set forth in SEQ ID NO:33, and the isolated antigen binding protein may comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:30 and the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:31. The isolated antigen binding protein is also referred to as 900428.

[0199] In the present application, the isolated antigen binding protein is capable of competing with a reference antibody for binding to the TIGIT protein, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57, and the light chain variable region of the reference antibody may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:65, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66.

[0200] In the present application, the isolated antigen binding protein is capable of competing with a reference antibody for binding to the TIGIT protein, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:4, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:5, wherein the light chain variable region of the reference antibody may comprise LCDR1, LCDR2 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8.

[0201] The proteins, polypeptides and / or amino acid sequences of the present application should also be understood to encompass at least the following: variants or homologs having the same or similar function as said proteins or polypeptides.

[0202] In the present application, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., an antigen binding protein as described herein). For example, the functional variant may consist of a protein or polypeptide that already has at least one, e.g., 1-30, 1-20 or 1-10, e.g., 1, 2, 3, 4 or 5, amino acid substitution, deletion and / or insertion amino acid changes. The functional variant may substantially retain the biological properties of the protein or polypeptide before it is altered (substitution, deletion, addition, etc.). For example, the functional variant may retain at least 60%, 70%, 80%, 90% or 100% of the biological activity of the protein or polypeptide before it is altered (e.g., ability to bind to an antigen). For example, the substitution may be a conservative substitution.

[0203] In the present application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to a corresponding amino acid sequence in an antibody of a particular species or may belong to a particular class. For example, both the variable and constant regions of the antibody may be derived from both the variable and constant regions of an antibody of an animal species (e.g., human). In the present application, the homologue may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or polypeptide (e.g., an isolated antigen-binding fragment thereof described herein).

[0204] In this application, homology is generally defined as the similarity, similarity or relatedness between two or more sequences. The "sequence homology rate" can be calculated by comparing two matching sequences in a comparison window, determining the number of positions where both sequences have the same nucleic acid base (e.g., A, T, C, G) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Glyn, Cys and Met), and calculating the number of matches. The number of matching positions is calculated from Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, Met), divided by the total number of positions in the comparison window (window size), and multiplied by 100 to calculate the sequence homology rate. Comparison to determine sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithms required to achieve maximum comparison within the full length sequence range or within the target sequence region to be compared. These homologies can also be measured by the FASTA and BLAST methods. For a description of the FASTA algorithm, see R. Pearson and DJ Lipman. "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci.), 85:2444-2448, 1988, and DJ Lipman and WR Pearson "Rapid and sensitive protein similarity searches", Science, 227:1435--1441, 1989. For a description of the BLAST algorithm, see R. Pearson and DJ Lipman. "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci.), 85:2444-2448, 1988, and DJ Lipman and WR Pearson "Rapid and sensitive protein similarity searches", Science, 227:1435--1441, 1989.S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman. "Basic local alignment search tool", Journal of Molecular Biology, 215:403-410, 1990.

[0205] The antigen-binding proteins (e.g., TIGIT antibodies) described herein are capable of specifically binding the TIGIT antigen. "Specific binding" An antigen-binding protein (e.g., an antibody) of a TIGIT antigen can usually bind to TIGIT with a KD value of about 1 nM or higher affinity (e.g., 1 nM, 100 pM, 10 pM, 2 pM, or 1 pM), but does not bind to other proteins that lack the TIGIT sequence. For example, "specific binding" an antibody against TIGIT does not bind to human CD226, human CD155, and human CD112. The antigen-binding proteins (e.g., antibodies) described herein specifically bind to the TIGIT antigen or its labeled form (e.g., fluorescently labeled TIGIT antigen), and do not bind to other proteins that lack the TIGIT epitope. Whether an antigen-binding protein (e.g., an antibody) binds to a TIGIT antigen can be determined using any assay known in the art. Examples of assays known in the art for determining binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET). In certain embodiments, the TIGIT antibodies described herein can also cross-react with monkey and / or mouse TIGIT, as detected, for example, by flow analysis techniques or enzyme-linked immunosorbent assays. As used herein, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.

[0206] The antigen-binding protein (e.g., TIGIT antibody) described in the present application inhibits the binding of TIGIT to CD155 ligand. The blocking test can be carried out, for example, by mixing the antigen-binding protein (e.g., TIGIT antibody) with an antigen (or a cell capable of expressing the antigen) and a ligand of the antigen (or a cell expressing the ligand), and reacting the ability of the antigen-binding protein to competitively bind to the ligand of the antigen based on the intensity (e.g., fluorescence intensity or concentration) of a detection marker. For example, the IC50 for blocking the binding of the TIGIT antigen and CD155 ligand by the antigen-binding protein (e.g., TIGIT antibody) described in the present application is about 0.1 μg / ml-0.05 μg / ml when detected using flow cytometry.

[0207] Nucleic acids, vectors, and cells

[0208] However, the present application also provides one or more isolated nucleic acid molecules. The one or more nucleic acid molecules may encode the antigen-binding protein described herein. For example, each of the one or more nucleic acid molecules may encode the complete antigen-binding protein or a portion thereof (e.g., one or more of HCDR1-3, LCDR1-3, VL, VH, light chain, or heavy chain).

[0209] The nucleic acid molecules described herein can be isolated, for example, produced or synthesized (i) in vitro, e.g., by polymerase chain reaction (PCR) amplification, (ii) by clonal recombination, (iii) by purification, e.g., by enzymatic digestion and stepwise separation by gel electrophoresis, or (iv) synthetically, e.g., chemically. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0210] In the present application, the nucleic acid encoding the antibody, its antigen-binding fragment can be prepared by various methods known in the art, including but not limited to overlap extension PCR using restriction fragment manipulation, or synthetic oligonucleotides (for details, see Sambrook et al., Molecular Cloning, A Laboratory, Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Inc. Wiley-Interscience, New York NY, 1993).

[0211] Meanwhile, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. The vector may also comprise other genes, such as marker genes, for selecting the vector under appropriate conditions in a suitable host cell. The vector may also comprise expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art, and for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatable element. The specific structure of the expression control sequence may vary depending on the species or cell type function, but may typically include 5' non-transcribed sequences involved in transcription and translation initiation, such as TATA, capping sequences, CAAT sequences, and 5' and 3' non-translated sequences. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence operably linked to a nucleic acid for transcriptional control. The expression control sequence may further include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters are, for example, promoters of SP6, T3 and T7 polymerase, human U6RNA promoter, CMV promoter and its artificial heterologous promoter (for example, CMV), a part of which may be fused to a part of the promoter of other cellular protein (for example, human GAPDH, glyceraldehyde 3-phosphate dehydrogenase) gene, which may or may not further include an intron. One or more nucleic acid molecules described in the present application may be operably linked to an expression control element. The vector may be, for example, a plasmid, a slime mold, a virus, a phage, or other vectors commonly used, for example, in genetic engineering. For example, the vector is an expression vector.

[0212] Meanwhile, the present application provides host cells, which may comprise one or more nucleic acid molecules and / or one or more vectors described herein. In some embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors described herein. In some embodiments, each or each host cell may comprise a plurality (e.g., two or more) or a plurality (e.g., two or more) of nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into the host cell, e.g., a eukaryotic cell, such as a plant, fungus or yeast cell. The vectors described herein may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0213] Preparation method

[0214] On the other hand, the present application provides a method for preparing said antigen-binding protein. The method may comprise culturing said host cell described herein under conditions allowing expression of said antigen-binding protein. This can be done, for example, by using an appropriate medium, an appropriate temperature and incubation time, etc., and is a method known to the skilled person.

[0215] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding proteins (e.g., anti-TIGIT antibodies) of the present application. For example, animals can be immunized with conjugated or naturally occurring TIGIT homodimers or fragments thereof. Any suitable immunization method can be used, such as adjuvants, immunostimulants, booster immunizations, etc., and one or more routes can be used.

[0216] Any suitable form of TIGIT can be used as an immunogen (antigen) to generate non-human antibodies specific to TIGIT, and the antibodies can be screened for biological activity. The stimulating immunogen can be full-length mature human TIGIT, including the natural homodimer, or a peptide containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen can be purified from natural sources or made in genetically modified cells. The DNA encoding the immunogen can be of genomic or non-genomic origin (e.g., cDNA). The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, plasmids, and non-viral vectors.

[0217] An exemplary method for producing the anti-human TIGIT antibodies of the present application is described in Example 1.

[0218] The humanized antibody can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA and IgE. In the present application, the antibody is an IgG antibody, and the IgG1 isotype is used. Optimization of the sequence of the required constant structural domain can be achieved by screening the antibody in the biological assay described in the following examples to obtain the desired biological activity. Again, either class of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof can be used in the compounds and methods herein.

[0219] An exemplary method for humanizing the anti-human TIGIT antibodies of the present application is described in Example 2.

[0220] The sequence of the DNA molecule of the antigen binding protein of the present application or a fragment thereof can be obtained by conventional techniques such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused to form a single chain antibody.

[0221] Once a sequence of interest is obtained, it can be obtained in large quantities by recombinant techniques, typically by cloning it into a vector, transplanting it into cells, and isolating the sequence of interest from the grown host cells by standard techniques.

[0222] It is also possible to synthesize sequences by artificial synthesis, especially when the length of the fragment is short. In many cases, very long fragments can be obtained by synthesizing several smaller fragments and ligating them together. This nucleic acid molecule can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art.

[0223] The present application also relates to vectors comprising suitable nucleic acid molecules as described above and suitable promoters or control sequences. These vectors can be used to transform suitable host cells so that they can express the protein. The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. For example, animal cells can include, but are not limited to, CHO-S, CHO-K1, HEK-293 cells, etc.

[0224] The steps described herein for transforming a host cell with recombinant DNA can be carried out by techniques well known in the art. The resulting transformant can be cultured in a conventional manner, and the transformant expresses the polypeptide encoded by the nucleic acid molecule of the present application. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium. Typically, the transformed host cell is cultured under conditions suitable for the expression of the antigen binding protein of the present application. The antigen binding protein of the present application can then be purified using conventional immunoglobulin purification steps such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, and conventional separation and purification means known to those skilled in the art.

[0225] The monoclonal antibodies obtained can be identified by routine methods, for example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays such as flow cytometric sorting (FACS), radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0226] Pharmaceutical Compositions

[0227] Further, the present application provides a composition. In certain embodiments, the composition may be a pharmaceutical composition comprising the antigen-binding protein of the present application or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell, and a pharma- ceutically acceptable vector. Typically, these substances may be formulated in a non-toxic, inert and pharma- ceutically acceptable aqueous vector vehicle, in which case the pH may typically be about 5-8, for example, about 6-8, and the pH may vary depending on the nature of the substances formulated and the condition to be treated. The formulated pharmaceutical composition may be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0228] The antigen-binding proteins described herein may also be cellular therapeutic agents expressed intracellularly by nucleotide sequences for such antibodies, e.g., for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0229] The pharmaceutical composition described in the present application can directly bind to the TIGIT protein molecule, and therefore can be used for the prevention and treatment of TIGIT-related diseases, and can also be used in combination with other therapeutic agents.

[0230] The pharmaceutical composition of the present application may contain a safe and effective amount of the antigen-binding protein described herein (e.g., 0.001-99 wt%, 0.01-90 wt%, or 0.1-80 wt%), and a pharma- ceutically acceptable adjuvant (which may include a carrier or excipient). Such vectors may include, but are not limited to, table salt, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The formulation must be adapted to the mode of administration. The pharmaceutical composition described herein may be in the form of an injection, for example, by preparing a saline solution or an aqueous solution containing glucose and other excipients in a conventional manner. Pharmaceutical compositions such as injections and solutions are desirably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, from about 1 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the antigen-binding protein described herein may be used in combination with other therapeutic agents.

[0231] The antigen binding proteins or pharmaceutical compositions described herein can be formulated, administered, and administered in a manner consistent with good medical practice. Considerations in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to the practitioner. The therapeutic agent (e.g., anti-TIGIT antibody) is optionally, but not necessarily, formulated and / or administered simultaneously with one or more agents currently used to prevent or treat the condition under consideration. The effective amount of such other agents will depend on the amount of therapeutic agent (e.g., anti-TIGIT antibody) present in the formulation, the type of condition or treatment, and other factors discussed above. These agents can generally be administered at any dose and by any route deemed empirically / clinically appropriate. The dose of the antibody administered in the combination therapy can be reduced compared to the individual treatments. This treatment can be easily monitored with conventional techniques.

[0232] Methods and Uses

[0233] Meanwhile, the present application provides for the use of said antigen binding protein, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the preparation of a medicament. The medicament is used for the prevention, alleviation and / or treatment of a TIGIT-associated disease. In certain embodiments, the described TIGIT-associated disease may be a T cell dysfunction disease. T cell dysfunction is reflected in T cell exhaustion, and the enhancement of NK cells and activation of T cells can enhance the body's immune activity to treat, delay or ameliorate the disease. For example, the TIGIT-associated disease may be a tumor, cancer or infectious disease. For example, the TIGIT-associated disease may be a CD155-positive or PVR-positive tumor, cancer, immune disease or infectious disease, among others. In certain embodiments, the tumor may be selected from the group consisting of breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, kidney cancer, melanoma, T lymphoma, pancreatic cancer. For example, the tumor is colon cancer. For example, the antigen binding proteins described herein inhibit tumor growth in animal models of tumors selected from the following group: breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, renal cancer, melanoma, T lymphoma, and pancreatic cancer.

[0234] The antigen-binding proteins of the present application can inhibit tumor growth and / or inhibit tumor cell proliferation. In some embodiments, the tumor comprises colon cancer. In some embodiments, the tumor or cancer is a tumor or cancer in which TIGIT expression is abnormal. The present application also provides a method for detecting TIGIT expression in a biological sample, which is described below. In certain embodiments, the method includes contacting a biological sample with an antigen-binding protein described herein under conditions in which the antigen-binding protein can bind to TIGIT, and detecting whether a complex is formed between the antigen-binding protein and TIGIT. For example, the tumor or cancer is a tumor or cancer in which TIGIT expression is elevated compared to a non-tumor or cancer sample. Such a method may be an in vitro method or an in vivo method. The antigen-binding proteins described herein can be used, for example, in immunoassays, including immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., protein blotting), flow cytometry (e.g., FAGS), and enzyme-linked immunosorbent assay (ELISA), etc. In certain embodiments, for example where TIGIT is a biomarker for patient selection, the antigen binding protein is used to select subjects suitable for treatment with the antigen binding protein and / or fusion protein described herein. The present application also provides for the use of the antigen binding protein in a method for diagnosing a subject suffering from a medical condition (e.g., cancer or immune dysfunction), comprising contacting a sample obtained from a subject with the antigen binding protein of the present application and detecting the presence of the bound antigen binding protein and / or fusion protein, thereby determining the presence or expression level of TIGIT in the sample.

[0235] For example, in a mouse model of colon cancer, an antigen binding protein described herein (eg, a TIGIT antibody) can slow tumor growth.

[0236] The antigen binding proteins of the present application can be used in assay applications, for example, to test a sample, thereby providing diagnostic information.

[0237] In the present application, the sample used includes cells, tissue samples, and biopsy samples. In the present application, the term "biopsy" is intended to include any type of biopsy known to those skilled in the art. Thus, a biopsy as used in the present application can consist of a tissue sample prepared, for example, by endoscopic methods, or by puncture or needle biopsy of an organ.

[0238] As used herein, a sample includes fixed or preserved cell or tissue samples.

[0239] The present application provides kits containing the antigen-binding proteins of the present application. In certain embodiments, the kits may include containers, instructions for use, buffers, etc. For example, the proto-binding proteins of the present application may be immobilized on an assay plate.

[0240] The following examples are not intended to be limited by theory, and are only used to explain the proteins, preparation methods, uses, etc. of the present application, and are not used to limit the scope of the present application. Experimental methods for which no specific conditions are given in the following examples are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are percentages and parts by weight.

[0241] Working Example

[0242] Example 1 Mouse monoclonal antibody against human TIGIT - Preparation of primary antibody

[0243] 1.1 Generation of hybridoma cells producing mouse monoclonal antibodies

[0244] The method for producing mouse-derived monoclonal antibodies was the hybridoma production technique invented by Kohler and Milstein in 1975 (Nature, 1975, 256: 495-497). After emulsifying mouse Fc tag protein (ACRO, # tit-h5253) of human TIGIT with Freund's adjuvant, five mice of each strain, BALB / c, CD1, and C57BL / 6, were subcutaneously immunized. After three immunizations, serum was collected and tested for potency by ELISA, binding activity and functional activity by FACS, and the optimal mice for fusion of spleen cells with SP2 / 0 myeloma cells were selected. After screening the hybridoma polyclonal cells with HAT, the polyclonal cell lines that could specifically bind to human TIGIT and block TIGIT-CD155 binding were screened for monoclonality by ELISA and FACS, and the monoclonal cell lines that specifically bind were screened again by ELISA and FACS to test their binding to monkey and mouse TIGIT. FACS screening of monoclonal antibodies that block the binding of TIGIT-CD155 and TIGIT-CD122 was performed, and then the cell function activity measurement and affinity (Biacore) screening of the screened monoclonal cell lines were performed to obtain monoclonal hybridoma cell lines expressing human TIGIT antibodies for sequence analysis. The screening data are shown in Table 1. [Table 1] TIFF0007627469000002.tif183170

[0245] As a result of high-throughput screening, we succeeded in obtaining the final monoclonal hybridoma cell lines with high affinity for both human and monkey binding activity, simultaneous blocking of the two ligands of TIGIT, CD155 and CD122, and biological activity. The hybridoma cell lines in Table 1 above are humanized.

[0246] Example 2 Cloning and humanization of the variable region gene sequence of anti-TIGIT antibody

[0247] 2.1 Cloning of antibody variable region genes in hybridoma cells

[0248] The cDNA sequences of the variable regions of the mouse antibodies expressed by the hybridoma cell lines were cloned based on the principle of TAKARA's 5'RACE technology. Briefly, heavy and light chain variable region gene-specific cDNAs were synthesized using the SMARTer 5' RACE synthesis experiment kit (TAKARA, #634859) according to the instructions. The 5' and 3' ends of the cDNA sequences were modified with PCR primers. The primers were designed to add appropriate lead sequences to the heavy and light chain variable region cDNAs, respectively, allowing the resulting PCR products to be seamlessly cloned into the existing recombinant antibody expression heavy chain vector pHB-Fc and light chain vector pHB-Cκ. The pHB-Fc expression vector contains the gene sequence of the human IgG1 heavy chain constant region with the L234A and L235A (Eu encoding) mutations on CH2 that have the antibody ADCC knockout (KO) effect, and the pHB-Cκ vector contains the gene sequence of the human κ light chain constant region. The heavy and light chain variable region PCR amplification products were cloned into expression vectors using In-fusion cloning reagent (TAKARA, #639650) and transformed into E. coli DH5α receptor cells (YB Biotech, #FYE607-80VL). Monoclonal colonies were selected for Sanger sequencing and analyzed to obtain the antibody variable region sequences. The sequences of the variable regions of the anti-TIGIT antibody expressed by B3 / 29F6 were as follows:

[0249] B3 / 29F6 VH SEQ ID NO:1 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWIWIRQFPGNKVEWMGYITSSGSTSYNPSLKSRISFTRDTSKNQFFLQLTSVTTEDTATYYCARLDFGNYGGAMDYWGQGTSVTVSS

[0250] B3 / 29F6 VL SEQ ID NO:2 DIVMTQSHKFMSTSIGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYITPYTFGGGTKLEIK

[0251] The underlined parts are CDRs (as defined by IMGT, the sequences are as follows): [Table 2]

[0252] 2.2 Construction of IgG1 wild-type chimeric expression vector

[0253] The IgG1 wild-type chimeric antibody and the ADCC KO chimeric antibody share the same light chain vector, with the difference being amino acids 234 and 235 of the heavy chain CH2 on the vector containing the human heavy chain constant region sequence, with the ADCC KO type being A234 / A235 and the wild type being L234 / L235. The IgG1 wild-type chimeric heavy chain expression vector was constructed in the same manner as described in 2.1. That is, the heavy chain variable region gene was amplified by PCR with a primer containing the lead sequence, and cloned by seamless ligation into a vector containing the human heavy chain constant region sequence (CH2 L234 / L235), completing the construction of the IgG1 wild-type chimeric expression vector.

[0254] 2.3 Expression of chimeric antibodies

[0255] The expression vectors obtained in 2.1 and 2.2 were amplified in E. coli, and an endotoxin-removed plasmid extraction kit (TianJian Biotech, #DP117) was used to prepare sufficient plasmid for transient transfection of the chimeric antibody. The host cells used for expression were CHO-S cells (Thermo Fisher, #R80007). CHO-S cells were transfected by mixing two types of heavy chain vectors and light chain vectors, which had been prepared separately, with polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes and culturing the cells in an incubator for 5 to 7 days. The cell culture supernatant was collected by centrifugation and purified with a protein A affinity chromatography column to obtain an ADCC KO-type human-mouse chimeric antibody (no. 900424) and an IgG1 wild-type human-mouse chimeric antibody (protein no. 900445).

[0256] According to the above method, the sequences of the chimeric antibodies VH and VH obtained are as follows:

[0257] 900423 and 900428 VH SEQ ID NO:32 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNRLEWMGYISYSGAPRYNPSLKSRISITRDTSKNQFFLQLSFVTTEDTATYYCASLGTDYYAMDYWGQGTSVIVSS

[0258] 900424 VH SEQ ID NO:1 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWIWIRQFPGNKVEWMGYITSSGSTSYNPSLKSRISFTRDTSKNQFFLQLTSVTTEDTATYYCARLDFGNYGGAMDYWGQGTSVTVSS

[0259] 900424 VL SEQ ID NO:2 DIVMTQSHKFMSTSIGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYITPYTFGGGTKLEIK

[0260] 900423 VL SEQ ID NO:34 DIVMTQSHKFMFTSVGDRVSITCKASQHVSNAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFIGRGSGTFTFTISSVQAEDLAVYYCQQYYSLPWTFGGGTKLEIK

[0261] 900428 VL SEQ ID NO:33 DIVMTQSHKFMFTSVGDRVSITCKASQHVSNAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGRGSGTDFTFTINSVQAEDLAVYYCQQYYSLPWTFGGGTKLEIK

[0262] Here, the amino acid sequence of the heavy chain constant region of 900424 is shown in SEQ ID NO: 30, the amino acid sequence of the heavy chain constant region of 900423 is shown in SEQ ID NO: 30, and the amino acid sequence of the heavy chain constant region of 900428 is shown in SEQ ID NO: 30. The light chain constant regions are all human kappa light chain constant regions (SEQ ID NO: 31).

[0263] 2.4 Humanization of mouse-derived anti-human TIGIT antibodies - Preparation of humanized antibodies

[0264] The following method was used for antibody humanization: the sequences of the antibody variable regions were compared with the available sequences in the NCBI IgBlast database, and finally, the human-derived conformational regions (FR regions) suitable for constructing CDR-grafted heavy and light chains on them were identified and analyzed.

[0265] The modification sites were designed according to the conserved and important amino acid residues in the FR region of human antibodies, humanization mutations were designed for the heavy and light chain variable regions of the chimeric antibody, and a humanized point mutant antibody expression plasmid was amplified and constructed using PCR technology. This humanized point mutant antibody expression plasmid was expressed in CHO-S cells and purified to obtain humanized antibody protein. The receptor binding ability, functional inhibitory activity and ADCC effect were screened using ELISA, Biacore and flow cytometry assays, and four types of humanized anti-TIGIT antibodies with excellent performance were obtained. The VH and VL sequences of the obtained humanized anti-TIGIT antibodies are shown below:

[0266] 900461 and 900464 VH SEQ ID NO:9 QVQLQESGPGLVKPSETLSLTTCTVSGYSITSDYAWIWIRQPPGKGLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS

[0267] 900461 VL SEQ ID NO:10 DIQMTQSPSSLSASVGDRVTITCRASQHVSTAVNWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK

[0268] 900464 and 900476 VL SEQ ID NO:11 DIQMTQSPSSLSASVGDRVTITCKASQHVSTALAWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK

[0269] 900466 VH SEQ ID NO:12 QVQLQESGPGLVKPSETLSLTTCTVSGYSITSDYAWIWIRQPPGKKLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS

[0270] 900466 VL SEQ ID NO:13 DIQMTQSPSSLSASVGDRVTITCKASQHVSTAVNWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK

[0271] 900476VH SEQ ID NO:14 QVQLQESGPGLVKPSETLSLTTCTVSGYSITSDYAWIWIRQPPGNKLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS

[0272] Here, the underlined parts are CDRs (as defined by IMGT), and 900461, 900464, 900466, and 900476 are the protein numbers of the four humanized antibodies corresponding to product numbers HB0030, HB0031, HB0032, and HB0033, respectively.

[0273] Here, the amino acid sequence of the heavy chain constant region of HB0030 is shown in SEQ ID NO: 29, the amino acid sequence of the heavy chain constant region of HB0031 is shown in SEQ ID NO: 29, the amino acid sequence of the heavy chain constant region of HB0032 is shown in SEQ ID NO: 29, and the amino acid sequence of the heavy chain constant region of HB0033 is shown in SEQ ID NO: 29. The light chain constant regions are all human kappa light chain constant regions (SEQ ID NO: 31).

[0274] Example 3 Assays for Chimeric and Humanized Antibodies

[0275] 3.1 Testing the binding activity of TIGIT antibodies to cells expressing human TIGIT

[0276] Chimeric antibodies 900324, 900423, 900424, 900428 and humanized antibodies HB0030, HB0031, HB0032, HB0033 were assayed for binding activity against cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Haber Bio).

[0277] All chimeric and humanized antibodies were diluted to 20 μg / ml in PBS containing 1% BSA (1% BSA / PBS) and added at 20 μL per well to a 96-well U-plate. A negative control (1% BSA / PBS only) was also set up at the same time. A suspension of human TIGIT-expressing cells (CHOK1-huTIGIT-2A3, Huabo Bio) in logarithmic growth phase was collected and centrifuged (1000 rpm x 5 min) to discard the culture medium, and the cells were incubated in 1% BSA / PBS at a viable cell density of 1 × 10 6 20 μL (2 × 10 / mL) was added per well of a 96-well U-plate together with anti-TIGIT antibody. 4 Cells) were added at room temperature for 30 min. The 96-well U-plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3 min) to discard the upper layer, washed once, and then PE-sheep anti-human-Fc (Jackson ImmunoResearch, #109-115-098) diluted 1:200 was added and incubated at room temperature for 15 min in the dark. The 96-well U-plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3 min) to discard the upper layer, washed three times in this manner, and finally resuspended in 100 μL of 1% BSA / PBS per well. The fluorescence intensity of the PE channel was measured using a flow cytometer (BD, #Canto II).

[0278] The results of the binding viability of the chimeric antibody are shown in Figure 1 and Table 3, and the results of the binding viability of the humanized antibody are shown in Figure 2 and Table 4. 900324 is a transient expression in CHO cells of the anti-human TIGIT antibody tiragolumab from Genentech, which has the same sequences of the light and heavy chain variable regions as the antibody 10A7 in patent application WO2015009856A2. As a result, it was confirmed that both the chimeric antibody and the humanized antibody have good and equivalent affinity for cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Haber Bio). [Table 3] [Table 4]

[0279] 3.2 Test to see whether TIGIT antibodies inhibit the binding of TIGIT antigen to TIGIT molecules (competitive method)

[0280] The antigen huCD155-moFc (ACROBiosystems, #CD5-H5254) was diluted to 40 μg / ml in PBS containing 1% BSA, and 10 μL was added to each well of a 96-well U-plate, and mixed thoroughly with anti-TIGIT antibody serially diluted at a volume ratio of 1:1. A positive control was also set up (CD155-moFc was added only). A suspension of human TIGIT-expressing cells (CHOK1-huTIGIT-2A3, Huabo Bio) in the logarithmic growth phase was removed by centrifugation (1000 rpm x 5 min), and the culture medium was diluted with 1% BSA / PBS to a viable cell density of 1 x 10 6 Resuspend at 2 × 10 / mL and place 20 μL (2 × 10 4The cells were added to the U-well plate at 100μL / well, mixed with CD155-moFc and anti-TIGIT antibodies for 30 minutes at room temperature, and culture was continued. The 96-well U-plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3min) to discard the upper layer, washed once, and then Alexa488-gat anti-mouse-Fc (Jackson ImmunoResearch, #115-545-071) diluted 1:300 was added and incubated at room temperature for 15 minutes in the dark. The U-well plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3min) to discard the upper layer, washed three times in this manner, and finally resuspended in 100μL of 1% BSA / PBS per well. Fluorescence intensity of the FITC channel was measured by flow cytometry (BD, #Canto II).

[0281] The results of the binding activity of the chimeric antibody are shown in Figure 3 and Table 5, and the results of the binding activity of the humanized antibody are shown in Figure 4 and Table 6. As a result, it was revealed that the chimeric antibody and the humanized antibody have the same blocking ability against human CD155 and exhibit significant blocking effect. [Table 5] [Table 6]

[0282] 3.3 Affinity testing of humanized monoclonal antibodies

[0283] In this test, the kinetics and affinity of antigen-antibody binding were measured using the SPR method. BIOCORE (GE, #Biacore 8K) was used. Sereis S Sensor Chip ProteinA chip (GE, #29-1275-56) was equilibrated at room temperature for 20 to 30 minutes, and the chip was set on the device. The antibody sample was diluted to the experimental working concentration with equilibration buffer and stored sealed at 2 to 8 °C. The antigen was diluted with equilibration buffer HBS-EP (10x) (GE, #BR-1006-69), and histidine tagged (His-Tag) human TIGIT protein (huTIGIT, Acro Biosystems, #TIT-H52H3), diluted antigen 20 nM was set at zero concentration twice in a concentration gradient starting from 2.5x dilution (※1). 1000 equilibration buffer) and duplicates of the lowest concentration were created. Glycine solution at pH 1.5 (GE, #BR100354) was used to regenerate the chip. The samples were analyzed using the capture multicycle kinetic program, and after confirming the absence of significant reference binding, the corresponding analysis program was selected, and the 1:1 binding model, Kinetics, was selected and fitted to the analysis to obtain the kinetic parameters of the samples.

[0284] The test results are shown in Figure 7. From the results of the affinity constant (KD(M)) using huTIGIT, the humanized monoclonal antibody HB0030 of the present application had an affinity of 10 -11 It was shown that the affinity was close to that of the control, and was extremely strong. [Table 7]

[0285] Example 4 In vivo efficacy studies in animals

[0286] In this study, the effects of anti-human TIGIT antibodies HB0030, HB0031, HB0032, and HB0033 were examined in immunodetection sites of humanized mice BALB / c-hPD1 / hTIGIT subcutaneously inoculated with the CT26.WT colon cancer model.

[0287] In this study, mouse colon cancer cells CT26.WT cells were harvested, and logarithmic growth phase CT26.WT cells were collected, the culture medium was removed, and the cells were washed twice with PBS before inoculation (CT26.WT cell viability was 98.57% and 98.28% before and after tumor, respectively). The inoculation dose was 5 × 10 5 / 100 μL / mouse. The average tumor volume was 94.02 mm on the 11th day after inoculation. 3 When the tumor volume reached 100 mm, the mice were randomly grouped according to tumor volume, with 8 mice in each group. The day of grouping was defined as D0, and the mice were administered on D0, D3, D7, D10, D14, and D17. After the start of administration, the tumor size was observed and the mice were weighed on D0, D2, D4, D6, D8, D10, D13, D15, D17, D20, and D23. Tumor volume calculation method: Tumor volume (mm 3 ) = 0.5 × tumor long axis × tumor short axis 2 The results are shown in Figure 5.

[0288] From the experimental results in FIG. 5, it is clear that the anti-TIGIT humanized monoclonal antibodies HB0030, HB0031, HB0032 and HB0033 of the present application have a significant tumor-suppressing effect when administered alone at 10 mg / kg.

[0289] Furthermore, we investigated the efficacy of HB0030 and the control antibody tiragolumab at 10 mg / kg in immunodetection site humanized mice BALB / c-hPD1 / hTIGIT subcutaneously inoculated with the CT26.WT colon cancer model.

[0290] In this test, CT26 cells were harvested from logarithmic growth phase mouse colon cancer cells, the culture medium was removed, and the cells were washed twice with PBS. Then, 5 × 10 5 The survival rates of CT26 cells before and after inoculation were 98.51% and 97.250%, respectively. The mean tumor volume was 80.33 mm at 12 days after inoculation. 3When the tumor volume reached 100 mm, the mice were randomly divided into groups according to tumor volume, with six mice in each group. The day of grouping was defined as D0, and the mice were administered the drugs on D0, D3, D7, D10, D14, and D17. After the start of administration, the tumor size was observed and the mice were weighed on D0, D2, D4, D6, D8, D10, D13, D15, D17, D20, and D23. Tumor volume calculation method: Tumor volume (mm 3 ) = 0.5 × tumor long axis × tumor short axis 2 The results are shown in Figure 6.

[0291] As can be seen from the results in FIG. 6, the anti-TIGIT antibody HB0030 of the present application exhibited a superior tumor-suppressing effect compared to the control antibody tiragolumab.

[0292] In this study, no significant changes in body weight were observed in any of the groups of mice treated 24 days after inoculation.

[0293] Example 5 In vivo efficacy test of anti-TIGIT antibody HB0030 in animals

[0294] To evaluate the role of ADCC effect of HB0030 molecule in vivo, 900541 is HB0030 with L234A / L235A mutation in Fc region with no ADCC effect, and the other two are samples of different batches of HB0030. In this experiment, saline was used as a negative control, and the antitumor effects of the above three drugs were evaluated on mouse colon cancer cells CT26.WT transplanted into humanized mice BALB / c-hPD1 / hTIGIT.

[0295] BALB / c-hPD1 / hTIGIT mice (inoculated with CT26.WT cells) were intraperitoneally administered 3 mg / kg of the test drug twice a week for a total of six doses.Compared with control G1 (saline), the tumor inhibition rates of 900541 (G2: anti-TIGIT-1, 3mPK) were 40.26% in TGItv and 32.60% in TGItw by tumor volume (TIGtv) and weight (TIGtw), and the tumor inhibition rates of anti-TIGIT antibody HB0030 (G3: anti-TIGIT-2, 3mPK) were 90.68% in TGItv and 86.85% in TGItw. Anti-TIGIT antibody HB0030 (G4: anti-TIGIT-3, 3mPK) significantly inhibited tumor growth by 82.38% in TGItv and 77.85% in TGItw (G3 vs. G1, ***Ptv<0.001, ***Ptw<0.001). Anti-TIGIT antibody HB0030 significantly inhibited tumor growth by 82% in TGItw (G4 vs. G1, ***Ptv<0.001, ***Ptw<0.001). There was no significant difference in tumor inhibition between (G 3: anti-TIGIT-2, 3mpk) and HB0030 (G 4: anti-TIGIT-3, 3mpk). (G4 VS G2, Ptv>0.263, Ptv>0.346)

[0296] Based on the above, when the test drug was intraperitoneally administered at 3 mg / kg twice a week for a total of six times to BALB / c-hPD1 / hTIGIT mice (inoculated with CT26.WT cells) in this test system, 900541 in G2 and HB0030 (3 mpk) in G3 and G4 showed clear tumor-suppressing effects compared to the saline control group.

Claims

1. An isolated antigen-binding protein comprising an antibody or antigen-binding fragment thereof, the isolated antigen binding protein is capable of binding to a TIGIT protein; containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have an amino acid sequence selected from the group consisting of 1) and 2) below:

4. The isolated antigen binding protein: 1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:4, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:8; or 2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

53.

2. The isolated antigen-binding protein of claim 1, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH and the light chain variable region VL comprise any one pair of amino acid sequences selected from the group consisting of: (1) VH: SEQ ID NO: 9, and VL: SEQ ID NO: 10, (2) VH: SEQ ID NO: 9, and VL: SEQ ID NO: 11, (3) VH: SEQ ID NO: 12, and VL: SEQ ID NO: 13, (4) VH: SEQ ID NO: 14, and VL: SEQ ID NO: 11, (5) VH: SEQ ID NO: 1, and VL: SEQ ID NO: 2, (6) VH: SEQ ID NO: 32, and VL: SEQ ID NO: 34, and (7) VH: SEQ ID NO: 32, and VL: SEQ ID NO:

33.

3. comprising a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises a human IgG heavy chain constant region, and the light chain constant region comprises a human Igκ constant region; 3. An isolated antigen binding protein according to claim 1 or 2.

4. 4. The isolated antigen binding protein of claim 3, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:

30.

5. 4. The isolated antigen binding protein of claim 3, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

31.

6. 6. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A polypeptide comprising the isolated antigen binding protein of any one of claims 1 to 5.

10. 10. An immunoconjugate comprising the isolated antigen-binding protein of any one of claims 1 to 5 or the polypeptide of claim 9.

11. 10. A method for preparing an isolated antigen binding protein according to any one of claims 1 to 5, comprising culturing a cell according to claim 8 under conditions which allow expression of the isolated antigen binding protein according to any one of claims 1 to 5.

12. 10. A pharmaceutical composition comprising an isolated antigen-binding protein according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a cell according to claim 8, a polypeptide according to claim 9 and / or an immunoconjugate according to claim 10, and optionally a pharma- ceutically acceptable adjuvant.

13. A pharmaceutical composition for the prevention, alleviation or treatment of a TIGIT-associated disease comprising an isolated antigen-binding protein according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a cell according to claim 8, a polypeptide according to claim 9, and / or an immune complex according to claim 10.

14. The pharmaceutical composition according to claim 13, wherein the TIGIT-related disease is a T cell dysfunction disease.

15. The pharmaceutical composition according to claim 13, wherein the TIGIT-related disease is a tumor.

16. The pharmaceutical composition of claim 15, wherein the tumor is a solid tumor.

17. The pharmaceutical composition of claim 15, wherein the tumor is colon cancer.

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