HK40033147B - Anti-tigit antibodies, preparation methods and use thereof - Google Patents

Description

Antibodies against TIGIT, preparation methods and uses thereof

Technical Field

The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting TIGIT, its preparation method and application.

Background Art

Under normal circumstances, T cells rely on the binding of exogenous or endogenous antigen-MHC-I/II complexes presented by antigen-presenting cells (APCs) to T cell (CD4 ⁺ , CD8 ⁺ ) surface receptors (T Cell Receptor, TCR) and additional co-stimulatory signals to fully activate, and then kill cells expressing relevant antigens through other effector cells. Conversely, T cells are also regulated by co-inhibitory signals, which have a negative regulatory effect on T cell activity, proliferation, survival, etc., and can also maintain self-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 mode, and its normalization is currently widely regarded as an important means of tumor treatment.

TIGIT (T cell Ig and ITIM domain, also known as WUCAM, Vstm3, or 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 the classic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoglobulin tyrosine-based tail (ITT) motifs. TIGIT is expressed in lymphocytes, with high expression in effector and regulatory CD4 ⁺ T cells, follicular helper CD4 ⁺ T cells, effector CD8 ⁺ T cells, and natural killer (NK) cells. The TIGIT gene is located on human chromosome 16 and encodes a 244-amino acid type I transmembrane protein. The human TIGIT molecule has a 141-amino acid extracellular domain with an immunoglobulin V-like domain; a 23-amino acid transmembrane domain; and a shorter 80-amino acid cytoplasmic domain with a PDZ-binding domain and an ITIM motif.

CD155 (also called PVR, Necl5 or Tage4) is a high-affinity ligand for TIGIT. Once CD155, which is highly expressed on the surface of tumors, binds to TIGIT on the surface of NK and T cells, their killing effect on tumor cells will be inhibited. TIGIT can inhibit the effect of NK cells by preventing the initial death of tumor cells and the release of tumor antigens; inhibit the co-stimulatory ability of dendritic cells, resulting in reduced cancer antigen presentation and increased anti-inflammatory cytokines such as IL-10. TIGIT can also induce PVR signaling in other cells such as tumor cells; TIGIT can directly inhibit the effect of CD8 ⁺ T cells, or TIGIT ⁺ Treg can inhibit CD8 ⁺ T cells to prevent the clearance of cancer cells (Manieri et al., Inhibition of the Cancer Immunity Cycle by TIGIT. 2016).

A large number of preclinical study results indicate that TIGIT will become a suitable target for patients with cancer and even other diseases. Therefore, given the role and function of TIGIT in various related diseases, there is still a need in this field to develop improved anti-TIGIT-specific antibodies suitable for treating patients.

Summary of the Invention

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 comprises the amino acid sequence shown in SEQ ID NO: 55. The isolated antigen-binding protein may also comprise at least one CDR in a light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 64. The isolated antigen-binding protein has one or more of the following properties: 1) capable of binding to TIGIT protein with a KD value of ^{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), wherein the KD value is determined by surface plasmon resonance; 2) capable of blocking the binding of CD155 to TIGIT in a FACS assay; and 3) capable of inhibiting tumor growth and/or tumor cell proliferation.

In one aspect, the present application provides an isolated antigen-binding protein comprising at least one CDR in a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:55.

In certain embodiments, the isolated antigen-binding protein comprises at least one CDR in the light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:64.

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

In certain embodiments, the isolated antigen binding protein comprises an antibody or an antigen binding fragment thereof.

In certain embodiments, the antigen binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.

In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

In certain embodiments, the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

In certain embodiments, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56.

In certain embodiments, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 4 and 42.

In certain embodiments, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57.

In certain embodiments, the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 5 and 43.

In certain embodiments, the VL comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:65.

In certain embodiments, the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 6 and 52.

In certain embodiments, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:7.

In certain embodiments, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:66.

In certain embodiments, the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 8 and 53.

In certain embodiments, the isolated antigen-binding protein competes with a reference antibody for binding to the TIGIT protein, wherein the reference antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, 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:56; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, and the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:65; 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:66.

In certain embodiments, the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

In certain embodiments, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:68.

In certain embodiments, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 15, 16, 44, and 45.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:70.

In certain embodiments, the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 17, 18, 46, and 47.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:72.

In certain embodiments, the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 19, 20, 21, 48, 49 and 50.

In certain embodiments, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:73.

In certain embodiments, the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 22 and 51.

In certain embodiments, the VL comprises the amino acid sequence shown in SEQ ID NO:64.

In certain embodiments, the VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 10, 11, 13, 33, and 34.

In certain embodiments, the isolated antigen-binding protein comprises an antibody light chain constant region, and the antibody light chain constant region is derived from a human κ light chain constant region, and the human κ light chain constant region comprises the amino acid sequence shown in SEQ ID NO:31.

In certain embodiments, the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:58.

In certain embodiments, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 23 and 35.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:60.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 25, 26, 36 and 37.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:61.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 27, 38, and 39.

In certain embodiments, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:62.

In certain embodiments, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 28, 40, and 41.

In certain embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:55.

In certain embodiments, the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 9, 12, 14, and 32.

In certain embodiments, the isolated antigen-binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a human IgG constant region.

In certain embodiments, the antibody heavy chain constant region comprises a human IgG1 constant region, and the human IgG1 constant region comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-30.

In another aspect, the present application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding protein.

On the other hand, the present application provides a vector comprising the nucleic acid molecule.

On the other hand, the present application provides a cell comprising the nucleic acid molecule or the vector.

On the other hand, the present application provides a method for preparing the isolated antigen-binding protein, which comprises culturing the cells under conditions that allow the isolated antigen-binding protein to be expressed.

On the other hand, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable adjuvant.

On the other hand, the present application provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and/or treating TIGIT-related diseases.

In certain embodiments, the TIGIT-related disease is a T cell dysfunction disorder.

In certain embodiments, the TIGIT-related disease is a tumor.

In certain embodiments, the tumor is selected from the group consisting of breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, kidney cancer, melanoma, T lymphoma, and pancreatic cancer.

On the other hand, the present application provides a method for inhibiting the binding of CD155 to TIGIT, which comprises administering the isolated antigen-binding protein.

On the other hand, the present application provides a method for preventing, alleviating or treating TIGIT-related diseases, which comprises administering the isolated antigen-binding protein to a subject in need thereof.

In certain embodiments, the TIGIT-related disease is a T cell dysfunction disorder.

In certain embodiments, the TIGIT-related disease is a tumor.

In certain embodiments, the tumor is selected from the group consisting of breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, kidney cancer, melanoma, T lymphoma, and pancreatic cancer.

Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

FIG1 shows the binding activity of TIGIT chimeric antibodies to cells expressing human TIGIT.

FIG2 shows the binding activity of humanized TIGIT antibodies to cells expressing human TIGIT.

FIG3 shows the blocking activity of TIGIT chimeric antibodies against the binding of human TIGIT to its ligand CD155.

FIG4 shows the blocking activity of TIGIT humanized antibodies on the binding of human TIGIT to its ligand CD155.

Figure 5 shows the efficacy test of TIGIT antibodies in animals.

Figure 6 shows the comparison of the efficacy of TIGIT antibodies and the control antibody Tiragolumab in animals.

DETAILED DESCRIPTION

The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

Definition of terms

In this application, the term "TIGIT" refers to "T cell immunoreceptor containing Ig and ITIM domains", which is generally a member of the PVR (poliovirus receptor) family of immunoglobulins, which can bind to PVR/CD155 and Nectin-2/CD112. The TIGIT of this application can refer to TIGIT proteins from any vertebrate source, including mammals, such as primates (e.g., humans, rhesus monkeys and crab-eating monkeys) and rodents (e.g., mice and rats). The term covers full-length TIGIT or fragments thereof (such as mature fragments lacking a signal peptide), unprocessed TIGIT, any form of TIGIT due to processing in cells, and artificially synthesized TIGIT. The term also covers variants of TIGIT, such as splice variants or allelic variants. In some cases, TIGIT is human TIGIT, and the amino acid sequence of human TIGIT comprises the amino acid sequence shown in UniProt accession number Q495A1. In some cases, TIGIT is cynomolgus monkey TIGIT, and the amino acid sequence of cynomolgus monkey TIGIT comprises the amino acid sequence set forth in UniProt Accession No. G7NXM4. In some cases, TIGIT is mouse TIGIT, and the amino acid sequence of mouse TIGIT comprises the amino acid sequence set forth in UniProt Accession No. P86176.

In this application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) ₂ , Fv fragment, 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, etc., as long as they exhibit the desired antigen-binding activity.

In this application, the term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain; 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) to the carboxyl terminus of the heavy chain CH1 domain; the term "F(ab') ₂ " generally refers to a dimer of Fab', an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fv" generally refers to the smallest antibody fragment containing a complete antigen recognition and binding site. In some cases, the fragment can be composed 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 the bond between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. In this application, the term "scFv" generally refers to a monovalent molecule formed by covalently linking one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have the general structure: NH2 _- VL-linker-VH-COOH or NH2 _- VH-linker-VL-COOH.

In this application, the term "antibody" is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies. An "antibody" can generally comprise a protein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs), which alternate with more conserved regions referred to as framework regions (FRs). Each VH and VL comprises three CDRs and four framework regions (FRs), arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of natural heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a β-sheet configuration, connected by three CDRs, forming loops connected, and in some cases forming part of the β-sheet structure. The CDRs in each chain are closely together through the FR region and, together with the CDRs from the other chain, form the antigen binding site of the antibody. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

In the present application, term " variable " generally refers to such fact, that is, some part of the sequence of the variable domain of antibody changes strongly, and it forms the combination and specificity of various specific antibodies to its specific antigen. However, variability is not evenly distributed in the whole variable region of antibody. It is concentrated in the three segments in light chain and heavy chain variable region, and is referred to as complementary determining region (CDR) or hypervariable region (HVR). The more highly conserved part in variable domain is referred to as framework (FR). In the art, the CDR of antibody can be defined by several methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Immunological Protein Sequence, 5th edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia definition rule based on structural loop region position (see, Al-Lazikani et al., J Mol Biol 273:927-48,1997) and the IMGT definition rule based on the concept of IMGT ontology (IMGT-ONTOLOGY) and IMGT Scientific chart rule.

IMGT refers to the International ImMunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http://www.imgt.org). IMGT specializes in immunoglobulins (IGs) or antibodies, T-cell receptors (TRs), major histocompatibility (MH) proteins from humans and other vertebrates, as well as the immunoglobulin superfamily (IgSF), MH superfamily (MhSF), and immune system-related proteins (RPIs) from vertebrates and invertebrates.

In this application, the term "isolated" antigen-binding protein generally refers to an antigen-binding protein that has been identified, separated and/or recovered from components of its production environment (e.g., natural or recombinant). Contaminating components of its production environment are generally substances that interfere with its research, diagnostic or therapeutic use and can include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. An isolated antigen-binding protein or antibody will generally be prepared by at least one purification step.

In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies in the cluster are identical, except for a small amount of natural mutations that may exist. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, different from conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on the antigen. Except for their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture and are not polluted by other immunoglobulins. The modifier "monoclonal" represents the feature of the antibody obtained from a substantially homogeneous antibody population, and is not to be construed as needing to produce antibodies by any ad hoc method. For example, the monoclonal antibody used herein can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., mouse-derived) antibody.

In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. In the CDR region, small additions, deletions, insertions, replacements, or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired properties, affinity, and/or ability. In some cases, the FR region residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

In this application, the term "fully human antibody" generally refers to antibodies expressed by animals by transferring human antibody-encoding genes into genetically engineered antibody gene-deficient animals. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

In this application, the terms "binding", "specific binding" or "specific for..." generally refer to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, which can determine the presence of a target in the presence of a heterogeneous population of molecules (including biological molecules). For example, an antibody binds to an epitope through its antigen binding domain, and this binding 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 an antibody that binds to this target with greater affinity, avidity, more readily and/or for a greater duration than it binds to other targets. An antibody is said to "specifically bind" to an antigen when it binds to an epitope more readily through its antigen binding domain than it would to a random, unrelated epitope. "Epitope" refers to a specific group of atoms (e.g., a sugar side chain, a phosphoryl group, a sulfonyl group) or an amino acid on an antigen to which an antigen binding protein (such as an antibody) binds.

In this application, the terms "KD" and " _KD " are used interchangeably and generally refer to the equilibrium dissociation constant. "KD" is 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 "binding rate (kon)" or "ka"). The association rate constant (kon), dissociation rate constant (kdis) and equilibrium dissociation constant (KD) can be used to express the binding affinity of an antigen-binding protein (e.g., an antibody) for an antigen. Methods for determining association and dissociation rate constants are well known in the art and include, but are not limited to, biomembrane interferometry (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction measured may be different if measured under different conditions (e.g., salt concentration, pH).

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

In this application, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" generally refers to a form of cytotoxicity in which secretory immunoglobulins bind to Fc receptors (FcRs) on certain cytotoxic effector cells (e.g., NK cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The primary cells mediating ADCC (e.g., NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII (see Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991)). In vitro and/or in vivo cytotoxicity assays can be performed to assess the ADCC activity of the target molecule, for example, an in vitro ADCC assay can be performed, as described in U.S. Patent No. 5,500,362 or No. 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells that can be used in such assays include PBMCs and NK cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model, such as that disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998). For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability.

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

In this application, the term "between" generally means that the C-terminus of a certain amino acid segment is directly or indirectly linked to the N-terminus of a first amino acid segment, and its N-terminus is directly or indirectly linked to the C-terminus of a second amino acid segment. In the light chain, for example, the N-terminus of L-FR2 is directly or indirectly linked to the C-terminus of LCDR1, and the C-terminus of L-FR2 is directly or indirectly linked to the N-terminus of LCDR2. For another example, the N-terminus of L-FR3 is directly or indirectly linked to the C-terminus of LCDR2, and the C-terminus of L-FR3 is directly or indirectly linked to the N-terminus of LCDR3. In the heavy chain, for example, the N-terminus of H-FR2 is directly or indirectly linked to the C-terminus of HCDR1, and the C-terminus of H-FR2 is directly or indirectly linked to the N-terminus of HCDR2. For another example, the N-terminus of H-FR3 is directly or indirectly linked to the C-terminus of HCDR2, and the C-terminus of H-FR3 is directly or indirectly linked to the N-terminus of HCDR3. In the present application, the "first amino acid fragment" and the "second amino acid fragment" can be any identical or different amino acid fragments.

In this application, the term "isolated" antigen-binding protein generally refers to an antigen-binding protein that has been identified, separated and/or recovered from components of its production environment (e.g., natural or recombinant). Contaminating components of its production environment are generally substances that interfere with its research, diagnostic or therapeutic use, and may include enzymes, hormones and other proteins or non-protein solutes. Isolated antigen-binding proteins or antibodies will generally be prepared by at least one purification step. The isolated antigen-binding proteins described herein generally do not bind to antigens other than TIGIT antigens.

In this application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" generally refers to DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, which is not associated with all or a portion of a polynucleotide found in nature, or linked to a polynucleotide to which it is not linked in nature.

In this application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and a vector primarily used for expression of the transcription and/or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

In this application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. The cell may include the progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but may be capable of expressing the antibody or antigen-binding fragment thereof described herein. The cell may be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia 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 NSO cell or a myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell. In this application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell, but also the offspring of these cells.

In this application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cells or mammals exposed thereto at the doses and concentrations employed. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™.

As used herein, the terms "administer" and "treat" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent or composition to an animal, human, subject, cell, tissue, organ or biological fluid. "Administer" and "treat" can refer to treatment, pharmacokinetics, diagnosis, research and experimental methods. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, and contact of a fluid with a cell. "Administer" and "treat" also mean in vitro and ex vivo treatment by an agent, a diagnostic, a binding composition or by another cell. "Treatment" when applied to a human, animal or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnosis; includes contact of a TIGIT binder with a human or animal, subject, cell, tissue, physiological compartment or physiological fluid.

As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including any TIGIT antigen binding protein and composition thereof of the present application, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent that effectively alleviates one or more symptoms of the disease (therapeutically effective amount). The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing (or destroying) cancer cell proliferation, reducing symptoms from the disease, improving the quality of life of individuals with the disease, reducing the dose of other drugs needed to treat the disease, delaying the progression of the disease, and/or prolonging individual survival.

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

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not.

In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 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 the specified value.

Detailed Description of the Invention

Antigen binding proteins

In one aspect, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR in the heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:55.

In one aspect, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR in the heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:54.

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

The antigen-binding protein described herein may comprise HCDR1, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3: GYSITSDYA. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen-binding protein described herein may comprise a HCDR2, wherein the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56: IX ₂ X ₃ SGX ₆ X ₇ , wherein X ₂ is S or T, X ₃ is S or Y, X ₆ is A or S, and X ₇ is P or T. For example, the sequence may be a sequence determined according to the IMGT definition rules.

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

The antigen-binding protein described herein may comprise HCDR2, wherein the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 4: ITSSGST. For example, the sequence may be a sequence determined according to the IMGT definition rules.

_The antigen-binding protein described herein may comprise a HCDR3, wherein the _HCDR3 may comprise the amino acid sequence set forth in _SEQ ID NO: ₅₇ : _{AX2LX4X5X6X7YX9X10AMDY} , wherein _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 X10} is G or absent. For example, the sequence may be a sequence defined according to the IMGT definition rules.

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

The antigen-binding protein described herein may comprise a HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 5: ARLDFGNYGGAMDY. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen binding protein described herein may further comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

The antigen-binding protein described herein may comprise a framework region H-FR1, wherein H-FR1 may comprise the amino acid sequence set forth in SEQ ID _NO : 58: _X1 VQLQESGPGLVKPSX16 _{X17 LSLTCTVX25} , wherein _X1 is D or Q, _X16 is E or Q, _X17 is S or T, and _X25 is S or T. For example, the sequence may be a sequence defined according to the IMGT definition rules.

For example, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 23 and 35.

The antigen-binding protein described herein may comprise a framework region H-FR1, wherein the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 23: QVQLQESGPGLVKPSETLSLTCTVS. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen-binding protein described herein may comprise a framework region H-FR2, wherein H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:60: WX ₂ WIRQX ₇ PGX ₁₀ X ₁₁ X ₁₂ EWX ₁₅ GY; wherein X ₂ is I or N, X ₇ is F or P, X ₁₀ is K or N, X ₁₁ is G, R or K, X ₁₂ is L or V, and X ₁₅ is I or M. For example, the sequence may be a sequence defined according to the IMGT definition rules.

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

The antigen-binding protein described herein may comprise a framework region H-FR2, wherein H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 59: WIWIRQPPGX ₁₀ X ₁₁ LEWIGY; wherein X ₁₀ is K or N, and X ₁₁ is G or K. For example, the sequence may be a sequence defined according to the IMGT definition rules.

For example, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 24-26.

_{The antigen-binding protein described herein may comprise a framework region H-FR3, wherein the H-FR3 may comprise the amino acid sequence of SEQ ID NO: 61: X1 YNPSLKSRX10 X11 X12 X13 X14 DTSKNQFX22 LX24 LX26 X27 VTX30 X31 DTATYYC , wherein X1 is R , S , or Y} , _X10 is I or V, _X11 is S or T, _X12 is F or I, X13 is S or T, _X14 is R or V, _X22 is F or S, _X24 is K or Q, _X26 is S or T, _X27 is F or S, _X30 is A or T, and _X31 is A or E. For example, the sequence may be a sequence determined according to the IMGT definition rules _.

For example, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 27, 38, and 39.

The antigen-binding protein described herein may comprise a framework region H-FR3, wherein the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 27: YYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen-binding protein described herein may comprise a framework region H-FR4, wherein the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 62: WGQGTX ₆ VX ₈ VSS, wherein X ₆ is L or S, and X ₈ is I or T. For example, the sequence may be a sequence determined according to the IMGT definition rules.

For example, the H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 28, 40, and 41.

The antigen-binding protein described herein may comprise a framework region H-FR4, wherein the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28: WGQGTLVTVSS. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen-binding protein described herein may comprise a light chain variable region VH, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 55: X ₁ VQLQESGPGLVKPSX ₁₆ X ₁₇ LSLTCTVX ₂₅ GYSITSDYAWX ₃₆ WIRQX ₄₁ PGX ₄₄ X _{4 5} X ₄₆ EWX 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 LX ₁₀₀ X ₁₀₁ X ₁₀₂ X ₁₀₃ YX ₁₀₅ X ₁₀₆ AMDYWGQGTX ₁₁₆ VX ₁₁₈ VSS, wherein X _{X 1} is D or Q, X ₁₆ is E or Q, X ₁₇ is S or T, X ₂₅ is S or T, X ₃₆ is I or N, X ₄₁ is F or P, X ₄₄ is N or K, X ₄₅ is G, K or R, X ₄₆ is L or V, X ₄₉ is I or M, X ₅₃ is S or T, X ₅₄ is S or Y, X ₅₇ is A or S, X ₅₈ is P or T, X ₅₉ is R, S or Y, X ₆₈ is I or V, X ₆₉ is S or T, X ₇₀ is F or I, X ₇₁ is S or T, X ₇₂ is R or V, X ₈₀ is F or S, X ₈₂ is K or Q, X ₈₄ is S or T, X ₈₅ is F or S, X ₈₈ is A or T, X ₈₉ is A or E, X ₉₈ is R or S, X _{X 100} is D or G, X ₁₀₁ is F or T, X ₁₀₂ is D or G, X ₁₀₃ is N or Y, X ₁₀₅ is G or absent, X ₁₀₆ is G or absent, X ₁₁₆ is L or S, and X ₁₁₈ is I or T. For example, the sequence can be a sequence determined according to the IMGT definition rules.

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: 1, 9, 12, 14 and 32.

The antigen-binding protein described herein may comprise a light chain variable region VH, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 54: QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX ₄₄ X ₄₅ LEWIGYITSSGSTYY NPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTV SS, wherein X ₄₄ is K or N, and X ₄₅ is G or K. For example, the sequence may be a sequence determined according to the IMGT definition rules.

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.

The antigen-binding proteins described herein may include a heavy chain constant region, which may be derived from human IgG, for example, human IgG1. In some cases, the Fc region of human IgG1 may be modified to achieve desired properties (e.g., ADCC KO). The modification may be an amino acid mutation. In some cases, the modification to IgG1 may be L234A/L235A, i.e., according to EU numbering, the amino acids at positions 234 and 235 are mutated from leucine (L) to alanine (A), respectively. In some cases, the heavy chain constant region of the antigen-binding proteins described herein may be derived from wild-type human IgG1.

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.

The isolated antigen-binding protein described in the present application may comprise at least one CDR in the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 64.

The isolated antigen-binding protein described in the present application may comprise at least one CDR in the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 63.

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

The antigen binding protein described herein may comprise LCDR1, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 65: QHVSX ₅ A, wherein X ₅ is N or T. For example, the sequence may be a sequence determined according to the IMGT definition rules.

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

The antigen binding protein described herein may comprise LCDR1, wherein the LCDR1 may comprise the amino acid sequence QHVSTA shown in SEQ ID NO: 6. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen binding protein described herein may comprise LCDR2, wherein the LCDR2 may comprise the amino acid sequence SAS shown in SEQ ID NO: 7. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen-binding protein described herein may comprise a LCDR3, wherein the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 66: QQX ₃ YX ₅ X ₆ PX ₈ T, wherein X ₃ is H or Y, X ₅ is I or S, X ₆ is L or T, and X ₈ is W or Y. For example, the sequence may be a sequence defined according to the IMGT definition rules.

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

The antigen binding protein described herein may comprise LCDR3, wherein the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 8: QQHYITPYT. For example, the sequence may be a sequence determined according to the IMGT definition rules.

The antigen binding protein described herein may further comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

The antigen-binding protein described herein may comprise a framework region L-FR1, wherein the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 68: DIX ₃ MTQSX ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ SX ₁₅ GDRVX ₂₀ ITCX ₂₄ AS; wherein X ₃ is Q or V, X ₈ is H or P, X ₉ is K or S, X ₁₀ is F or S, X ₁₁ is L or M, X ₁₂ is F or S, X ₁₃ is A or T, X ₁₅ is I or V, X ₂₀ is S or T, and X ₂₄ is K or R. For example, the sequence may be a sequence determined according to the IMGT definition rules.

For example, the L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 15, 16, 44 and 45.

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

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

The antigen-binding protein described herein may comprise a framework region L-FR2, wherein the L- _FR2 may comprise the amino acid sequence set _forth in _SEQ ID _NO :70: _{X1X2WYQQKPGX10X11PKLLIX17} ; wherein _X1 is L or V, _X2 is A or N, _X10 is K or Q, _X11 is A or S, and _X17 is H or Y. For example, the sequence may be a sequence defined according to the IMGT definition rules.

For example, the L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 17, 18, 46 and 47.

The antigen-binding protein described herein may comprise a framework region L-FR2, wherein the L-FR2 may comprise the _amino acid sequence set forth in SEQ ID NO: 69: _{X1X2WYQQKPGKAPKLLIY} ; wherein _X1 is L or V, and _X2 is A or N. For example, the sequence may be a sequence defined according to the IMGT definition rules.

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

The antigen-binding protein described herein may comprise a framework region L-FR3, wherein the L-FR3 may comprise the amino acid sequence of SEQ ID NO: 72: YX ₂ X ₃ X ₄ GVPX8RFX ₁₁ GX ₁₃ X ₁₄ SGTDFTX ₂₁ TIX ₂₄ SX ₂₆ QX ₂₈ EDX ₃₁ AX ₃₃ YYC; wherein X ₂ is L or R, X ₃ is Q or Y, X ₄ is S or T, X ₈ is D or S, X ₁₁ is I, S or T, X ₁₃ is R or S, X ₁₄ is G or R, X ₂₁ is F or L, X ₂₄ is N or S, X ₂₆ is L or V, X ₂₈ is A or P, X ₃₃ is F or L, and X ₁₄ is T or V. For example, the sequence may be a sequence determined according to the IMGT definition rules.

For example, the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 19-21 and 48-50.

The antigen-binding protein described herein may comprise a framework region L-FR3, wherein the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 71: YX ₂ X ₃ SGVPSRFSGSX ₁₄ SGTDFTLTISSLQPEDFATYYC; wherein X ₂ is L or R, X ₃ is Q or Y, and X ₁₄ is G or R. For example, the sequence may be a sequence determined according to the IMGT definition rules.

For example, the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 19-21.

The antigen-binding protein described herein may comprise a framework region L-FR4, wherein the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 73: FGX ₃ GTKLEIK, wherein X ₃ is G or Q. For example, the sequence may be a sequence determined according to the IMGT definition rules.

For example, the L-FR4 may include the amino acid sequence shown in any one of SEQ ID NOs: 22 and 51.

The antigen-binding protein described herein may comprise a framework region L-FR4, wherein the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 22: FGQGTKLEIK. For example, the sequence may be a sequence determined according to the IMGT definition rules.

₈ X 9 X ₁₀ X ₁₁ X 12 X1 3 SX 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 YYCQQX 91 YX 93 X 94 PX 96 TFGX 100 GTKLEIK , wherein X 3 is Q or V , X 8 is H or P , X 9 is K} or _{S , and} X or _S , and _X11 is L or M, _X12 is F or S, _X13 is A or T, _X15 is I or V, _X20 is S or T, _X24 is K or R, _X31 is N or T, _X33 is L or V, _X34 is A or N, _X42 is K or Q, _X43 is A or S, _X49 is H or Y, _X54 is L or R, _X55 is Q or Y, _X56 is S or T, _X60 is D or S, _X63 is I, S or T, _X65 is S or R, _X66 is G or R, X73 is L or F, _X76 is _S or N, _X78 is L or V, _X80 is A or P, _X83 is F or L, _X85 is T or V, X _{X 91} is H or Y, X ₉₃ is I or S, X ₉₄ is L or T, X ₉₆ is W or Y, and X ₁₀₀ is Q or G. For example, the sequence may be a sequence determined according to the IMGT definition rules.

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: 2, 10, 11, 13, 33 and 34.

The antigen-binding protein described herein may comprise a light chain variable region VL, wherein the VL may comprise the amino acid sequence set forth in SEQ ID NO: 63: DIQMTQSPSSLSASVGDRVTITCX ₂₄ ASQHVSTAX ₃₃ X ₃₄ WYQQKPGKAPKLLIYSASYX ₅₄ X ₅₅ SGVPSRFSGSX ₆₆ SGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK, wherein X ₂₄ is K or R, X ₃₃ is L or V, X ₃₄ is A or N, X ₅₄ is L or R, X ₅₅ is Q or Y, and X ₆₆ is G or R. For example, the sequence may be a sequence determined according to the IMGT definition rules.

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.

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, 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.

In the present application, the isolated antigen-binding protein may comprise the antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, 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.

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

In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain variable region CDR, namely HCDR1, HCDR2 and HCDR3, and the HCDR1, the HCDR2 and the HCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

In the present application, the isolated antigen-binding protein may comprise the antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, 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.

For example, in the present application, the isolated antigen-binding protein may comprise the antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 6 and 52, 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 any one of SEQ ID NOs: 8 and 53.

In the present application, the isolated antigen-binding protein may comprise antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, 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, 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.

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

In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

In certain cases, the isolated antigen-binding protein described herein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 7 and SEQ ID NO: 53.

In the present application, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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.

In certain cases, 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 any one of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 32 and SEQ ID NO: 14, and the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 13.

In the present application, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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.

In certain cases, 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 any one of SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:14, and the VL may comprise the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:13.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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.

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.

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.

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.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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.

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.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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.

The isolated antigen-binding protein described herein may further comprise a heavy chain constant region and a light chain constant region. The heavy chain constant region may be derived from the constant region of human IgG1, and the light chain constant region may be derived from the human κ light chain constant region.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:29, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO:31, and the isolated antigen-binding protein may be referred to as HB0030.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31. The isolated antigen-binding protein may be referred to as HB0031.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen-binding protein may be referred to as HB0032.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen-binding protein may be referred to as HB0033.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 30, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31. The isolated antigen-binding protein may be referred to as 900424.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 30, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen-binding protein may be referred to as 900423.

For example, the isolated antigen-binding protein may comprise a heavy chain variable region VH and a light chain variable region VL, 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; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 30, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen-binding protein may be referred to as 900428.

The isolated antigen-binding protein described in the present application can compete 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, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, 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; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57, the light chain variable region of the reference antibody may comprise LCDR1, LCDR2 and LCDR3, 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; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 66.

The isolated antigen-binding protein described in the present application can compete 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, the heavy chain variable region of the reference antibody may comprise HCDR1, HCDR2 and HCDR3, 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; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:5, the light chain variable region of the reference antibody may comprise LCDR1, LCDR2 and LCDR3, 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; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8.

The proteins, polypeptides and/or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

In the present application, the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., the antigen-binding protein described in the present application). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, such as 1-30, 1-20 or 1-10, or for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

In the present application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a particular species, or may belong to a particular class. For example, the variable region and constant portion of an antibody may both be from the variable region and constant region of an antibody from an animal species (e.g., human). In the present application, the homolog 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 to the amino acid sequence of the protein and/or polypeptide (e.g., the antigen-binding protein described herein).

In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined using the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

The antigen-binding proteins (e.g., TIGIT antibodies) described herein are capable of specifically binding to the TIGIT antigen. Antigen-binding proteins (e.g., antibodies) that "specifically bind" to the TIGIT antigen can typically 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 do not bind to other proteins that lack the TIGIT sequence. For example, antibodies that "specifically bind" to TIGIT do not bind to human CD226, human CD155, and human CD112. The antigen-binding proteins (e.g., antibodies) described herein are capable of specifically binding to the TIGIT antigen or a labeled form thereof (e.g., a fluorescently labeled TIGIT antigen), but do not bind to other proteins that lack the TIGIT epitope. Whether an antigen-binding protein (e.g., an antibody) binds to the 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 some cases, the TIGIT antibodies described herein may also cross-react with monkey and/or mouse TIGIT, for example, as detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA). As used herein, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.

The antigen binding protein described herein (e.g., TIGIT antibody) can inhibit the binding of TIGIT to the CD155 ligand. Blocking experiments can be detected using a competitive method, for example, the antigen binding protein (e.g., TIGIT antibody) is mixed with an antigen (or a cell that can express an antigen) and a ligand of the antigen (or a cell that expresses a ligand), and the ability of the antigen binding protein to competitively bind to the antigen is determined according to the intensity (e.g., fluorescence intensity or concentration) of the detectable marker. For example, as detected using a flow cytometer, the IC50 of the antigen binding protein described herein (e.g., TIGIT antibody) to block the binding of the TIGIT antigen to the CD155 ligand is about 0.1 μg/ml-0.05 μg/ml.

Nucleic acids, vectors, and cells

In another aspect, the application also provides one or more nucleic acid molecules that are isolated. The one or more nucleic acid molecules can encode the antigen-binding proteins described herein. For example, each of the one or more nucleic acid molecules can 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).

The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

In the present application, nucleic acids encoding the antibodies and antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific operations, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.

In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vectors may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Furthermore, the vectors may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequences are adjustable elements. The specific structure of the expression control sequences may vary depending on the function of the species or cell type, but typically include 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence functionally linked to a nucleic acid for transcriptional control. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a certain portion of the promoter may be fused to a certain portion of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene promoters, which may or may not include other introns. One or more nucleic acid molecules described herein may be operably connected to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

In another aspect, the application provides a host cell, which may comprise one or more nucleic acid molecules and/or one or more vectors described herein. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality of (e.g., 2 or more) or a variety of (e.g., 2 or more) nucleic acid molecules or vectors described herein. For example, the vector described herein may be introduced into the host cell, such as a eukaryotic cell, such as a cell from a plant, a fungus or yeast cell, etc. The vector described herein may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

Preparation method

In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may include culturing the host cell described herein under conditions that allow expression of the antigen-binding protein. For example, the method may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins (e.g., anti-TIGIT antibodies) of the present invention. For example, animals can be immunized with linked or naturally occurring TIGIT homodimers or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more approaches can be used.

Any suitable form of TIGIT can be used as an immunogen (antigen) to produce non-human antibodies specific for TIGIT and screen the biological activity of the antibodies. The stimulating immunogen can be full-length mature human TIGIT, including natural homodimers, or peptides containing single/multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, plasmids, and non-viral vectors.

An exemplary method for producing the anti-human TIGIT antibody of the present application is described in Example 1.

Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody is an IgG antibody, using the IgG1 subtype. Optimization of the necessary constant domain sequences can be achieved by screening the antibodies using the biological assays described in the Examples below to produce the desired biological activity. Similarly, any class of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains, or variants thereof can be used in the compounds and methods herein.

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

The sequence of the DNA molecule of the antigen-binding protein or its fragment of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. The nucleic acid molecule can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

The present application also relates to vectors comprising the above-mentioned appropriate nucleic acid molecules and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The host cells can be prokaryotic cells, such as bacterial cells; 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, and HEK-293 cells.

The steps of transforming host cells with recombinant DNA described in this application can be carried out using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of this application. Depending on the host cell used, conventional culture medium is used to culture under appropriate conditions. Generally, the host cells obtained by transformation are cultured under conditions suitable for expression of the antigen-binding proteins of this application. The antigen-binding proteins of this 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, which are well known to those skilled in the art.

The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as flow cytometry (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

Pharmaceutical composition

On the other hand, the present application also provides a composition. In some cases, the composition may be a pharmaceutical composition containing the antigen binding protein or its fusion protein or its ADC or corresponding CAR-T cells of the present application, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH can generally be about 5-8, for example, the pH can be about 6-8, and the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

The antigen binding protein described in the present application can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition described herein can be used to directly bind to TIGIT protein molecules and thus can be used to prevent and treat TIGIT-related diseases. In addition, other therapeutic agents can also be used simultaneously.

The pharmaceutical composition of the present application may contain a safe and effective amount (such as 0.001-99wt%, 0.01-90wt%, or 0.1-80wt%) of the antigen-binding protein described in the present application and a pharmaceutically acceptable adjuvant (which may include a carrier or excipient). Such carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition described in the present application can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the antigen-binding protein described in the present application can also be used with other therapeutic agents.

The antigen binding proteins or pharmaceutical compositions described herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Considerations in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of the condition, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners. The therapeutic agent (e.g., anti-TIGIT antibody) need not be, but is optionally 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 depends on the amount of the therapeutic agent (e.g., anti-TIGIT antibody) present in the formulation, the type of condition or treatment, and the other factors discussed above. These agents can generally be used in any dose determined empirically/clinically to be appropriate and by any route determined empirically/clinically to be appropriate. The dose of the antibody administered in the combination therapy can be reduced compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.

Methods and uses

On the other hand, the present application provides the use of the antigen binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug. The drug is used to prevent, alleviate and/or treat TIGIT-related diseases. In some cases, the TIGIT-related disease may be a T cell dysfunction disorder. T cell dysfunction is manifested in T cell exhaustion, and the treatment, delay or alleviation of the disease is achieved by enhancing NK cells and activating T cells and enhancing the body's immune activity. For example, the TIGIT-related disease may be a tumor, cancer or infectious disease. For example, the TIGIT-related disease may be a CD155-positive or PVR-positive tumor, cancer, immune disease or infectious disease, including tumors, cancer, immune diseases or infectious diseases, etc. In some cases, the tumor may be selected from the following group: 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 can inhibit tumor growth in a tumor animal model selected from the group consisting of breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, kidney cancer, melanoma, T lymphoma, and pancreatic cancer.

The antigen-binding proteins of the present application can inhibit tumor growth and/or inhibit tumor cell proliferation. In certain embodiments, the tumor comprises colon cancer. In certain 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 described below. In some cases, the method comprises contacting the biological sample with the antigen-binding protein described herein under conditions that allow the antigen-binding protein to 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 methods can be in vitro or in vivo methods. The antigen-binding proteins described herein can be used, for example, in immunoassays, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (e.g., Western blot), flow cytometry (e.g., FAGS), and enzyme-linked immunosorbent assay (ELISA). In certain cases, for example, when TIGIT is a biomarker for selecting a patient, the antigen-binding protein is used to select a subject suitable for therapy with the antigen-binding protein and/or fusion protein described herein. The present application also provides the use of the antigen binding protein in a method for diagnosing a subject suffering from a disease (e.g., cancer or immune dysfunction), the method comprising: determining the presence or expression level of TIGIT in a sample obtained from the subject by contacting the sample with the antigen binding protein of the present application and detecting the presence of the bound antigen binding protein and/or fusion protein.

For example, in a mouse model of colon cancer, the antigen binding proteins described herein (eg, TIGIT antibodies) can slow tumor growth.

The antigen binding protein of the present application can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.

In the present application, the sample (specimen) adopted includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present application should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present application can include tissue samples prepared, for example, by endoscopic methods or puncture or needle biopsy of an organ.

As used herein, samples include fixed or preserved cell or tissue samples.

The present application also provides a kit containing the antigen-binding protein of the present application. In some cases, the kit may further include a container, instructions for use, a buffer, etc. For example, the original binding protein of the present application may be fixed to a detection plate.

This application also provides the following implementation scheme:

1. An isolated antigen-binding protein comprising at least one CDR in a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 55.

2. The isolated antigen-binding protein according to embodiment 1, comprising at least one CDR in the light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 64.

3. The isolated antigen binding protein according to any one of embodiments 1-2, which has one or more of the following properties:

1) capable of binding to TIGIT protein with a KD value of 1× ^10-10 M or less, wherein the KD value is determined by surface plasmon resonance;

2) able to block the binding of CD155 to TIGIT in a FACS assay; and

3) Ability to inhibit tumor growth and/or tumor cell proliferation.

4. An isolated antigen binding protein according to any one of embodiments 1-3, which comprises an antibody or an antigen binding fragment thereof.

5. The isolated antigen binding protein of embodiment 4, wherein the antigen binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb.

6. The isolated antigen binding protein of any one of embodiments 4-5, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

7. The isolated antigen-binding protein according to any one of embodiments 1-6, wherein the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.

8. The isolated antigen-binding protein of embodiment 7, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 56.

9. The isolated antigen binding protein of any one of embodiments 7-8, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 4 and 42.

10. The isolated antigen binding protein of any one of embodiments 7-9, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57.

11. The isolated antigen binding protein of any one of embodiments 7-10, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5 and 43.

12. An isolated antigen binding protein according to any one of embodiments 2-11, wherein the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:65.

13. The isolated antigen-binding protein according to embodiment 12, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 6 and 52.

14. The isolated antigen binding protein of embodiments 12-13, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7.

15. The isolated antigen binding protein of any one of embodiments 12-14, wherein the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 66.

16. The isolated antigen binding protein of any one of embodiments 12-15, wherein the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 8 and 53.

17. An isolated antigen-binding protein according to any one of embodiments 3-16, which competes with a reference antibody for binding to the TIGIT protein, wherein the reference antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, 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:56; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, and the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:65; 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:66.

18. An isolated antigen binding protein according to any one of embodiments 2-17, wherein the VL comprises framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

19. The isolated antigen-binding protein of embodiment 18, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 68.

20. The isolated antigen binding protein of any one of embodiments 18-19, wherein the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 15, 16, 44, and 45.

21. The isolated antigen binding protein of any one of embodiments 18-20, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 70.

22. The isolated antigen binding protein of any one of embodiments 18-21, wherein the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 17, 18, 46, and 47.

23. The isolated antigen binding protein of any one of embodiments 18-22, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 72.

24. The isolated antigen binding protein of any one of embodiments 18-23, wherein the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 19, 20, 21, 48, 49, and 50.

25. The isolated antigen binding protein of any one of embodiments 18-24, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 73.

26. The isolated antigen binding protein according to any one of embodiments 18-25, wherein the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 22 and 51.

27. An isolated antigen binding protein according to any one of embodiments 2-26, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 64.

28. An isolated antigen binding protein according to any one of embodiments 2-27, wherein the VL comprises the amino acid sequence shown in any one of SEQ ID NO: 2, 10, 11, 13, 33 and 34.

29. An isolated antigen-binding protein according to any one of embodiments 1-28, which comprises an antibody light chain constant region, and the antibody light chain constant region is derived from a human κ light chain constant region, and the human κ light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 31.

30. The isolated antigen binding protein of any one of embodiments 1-29, wherein the VH comprises framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

31. The isolated antigen-binding protein of embodiment 30, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 58.

32. The isolated antigen binding protein of any one of embodiments 30-31, wherein the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 23 and 35.

33. The isolated antigen binding protein of any one of embodiments 30-32, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 60.

34. An isolated antigen binding protein according to any one of embodiments 30-33, wherein the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 24-26, 36 and 37.

35. The isolated antigen binding protein of any one of embodiments 30-34, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 61.

36. The isolated antigen binding protein of any one of embodiments 30-35, wherein the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 27, 38, and 39.

37. An isolated antigen binding protein according to any one of embodiments 30-36, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 62.

38. An isolated antigen binding protein according to any one of embodiments 30-37, wherein the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 28, 40 and 41.

39. An isolated antigen-binding protein according to any one of embodiments 1-38, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:55.

40. An isolated antigen binding protein according to any one of embodiments 1-39, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 9, 12, 14 and 32.

41. The isolated antigen binding protein of any one of embodiments 1-40, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region comprises a human IgG constant region.

42. An isolated antigen-binding protein according to any one of embodiments 1-41, wherein the antibody heavy chain constant region comprises a human IgG1 constant region, and the human IgG1 constant region comprises the amino acid sequence shown in any one of SEQ ID NOs: 29-30.

43. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of embodiments 1-42.

44. A vector comprising the nucleic acid molecule according to embodiment 43.

45. A cell comprising the nucleic acid molecule according to embodiment 43 or the vector according to embodiment 44.

46. A method of making the isolated antigen binding protein of any one of embodiments 1-42, the method comprising culturing the cell of embodiment 45 under conditions such that the isolated antigen binding protein of any one of embodiments 1-42 is expressed.

47. A pharmaceutical composition comprising the isolated antigen binding protein of any one of embodiments 1-42, the nucleic acid molecule of embodiment 43, the vector of embodiment 44 and/or the cell of embodiment 45, and optionally a pharmaceutically acceptable adjuvant.

48. Use of the isolated antigen-binding protein described in any one of embodiments 1-42, the nucleic acid molecule described in embodiment 43, the vector described in embodiment 44, the cell described in embodiment 45 and/or the pharmaceutical composition described in embodiment 47 in the preparation of a drug for preventing, alleviating and/or treating TIGIT-related diseases.

49. The use according to embodiment 48, wherein the TIGIT-related disease is a T cell dysfunction disorder.

50. The use according to any one of embodiments 48-49, wherein the TIGIT-related disease is a tumor.

51. The use according to embodiment 50, wherein the tumor is colon cancer.

52. A method of inhibiting the binding of CD155 to TIGIT, the method comprising administering the isolated antigen binding protein of any one of embodiments 1-42.

53. A method for preventing, alleviating or treating a tumor, the method comprising administering the isolated antigen binding protein of any one of embodiments 1-42 to a subject in need thereof.

Without intending to be bound by any theory, the following examples are merely illustrative of the antigen-binding proteins, preparation methods, and uses of the present application and are not intended to limit the scope of this application. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

Example

Example 1 Preparation of Mouse Monoclonal Antibody Against Human TIGIT - Primary Antibody

1.1 Preparation of hybridoma cells producing mouse monoclonal antibodies

The method for preparing mouse monoclonal antibodies utilizes the hybridoma preparation technique developed by Kohler and Milstein in 1975 (Nature, 1975, 256:495-497). Human TIGIT protein with a mouse Fc tag (ACRO, #TIT-H5253) was emulsified with Freund's adjuvant. Five mice from each of the BALB/c, CD1, and C57BL/6 strains were then subcutaneously immunized at multiple sites. After three rounds of immunization, serum was collected and assayed for titer by ELISA, and binding and functional activity by FACS. Splenocytes from the best mice were then fused with SP2/0 myeloma cells. After HAT screening of hybridoma polyclonal cells, ELISA and FACS methods were used to screen out polyclonal cell lines that specifically bind to human TIGIT and can block TIGIT-CD155 binding, and then monoclonalization was performed. ELISA and FACS methods were used again to screen out specifically binding monoclonal cell lines, and the binding ability to monkey and mouse TIGIT was detected. Monoclonal antibodies that block the binding of TIGIT-CD155 and TIGIT-CD122 were screened out by FACS. Subsequently, cell functional activity was tested, and the selected monoclonal cell lines were affinity screened (Biacore). Finally, monoclonal hybridoma cell lines expressing human TIGIT antibodies were obtained for sequence analysis. The screening data are listed in Table 1.

Table 1. Hybridoma screening data

After high-throughput screening, the resulting monoclonal hybridoma cell line has high affinity, can bind to both humans and monkeys, has blocking function against both TIGIT ligands CD155 and CD122, and has biological activity. The hybridoma cell lines listed in Table 1 above were humanized.

Example 2 Cloning and humanization of anti-TIGIT antibody variable region gene sequences

2.1 Cloning of variable region genes of antibodies in hybridoma cells

Based on the principles of TAKARA's 5'RACE technology, the cDNA sequences of the mouse antibody variable regions expressed by hybridoma cell lines were cloned. Briefly, the SMARTer 5'RACE Synthesis Kit (TAKARA, #634859) was used to synthesize the variable region gene-specific cDNAs of the heavy and light chains according to the instructions. The 5' and 3' ends of the cDNA sequences were modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, so that the resulting PCR products could be cloned into the existing recombinant antibody expression heavy chain vector pHB-Fc and light chain vector pHB-Cκ via seamless cloning. The pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence, in which the _CH2 carries the L234A and L235A (Eu encoding) mutations that cause antibody ADCC knockout (KO) effects; the pHB-Cκ vector contains the human κ light chain constant region gene sequence. The heavy and light chain variable region PCR amplification products were cloned into expression vectors using In-fusion cloning reagents (TAKARA, #639650) and transformed into E. coli DH5α competent cells (Yisheng Biotechnology, #FYE607-80VL). Monoclonal colonies were selected for Sanger sequencing, and the antibody variable region sequences were obtained. The variable region sequence of the anti-TIGIT antibody expressed by B3/29F6 is as follows:

B3/29F6 VH SEQ ID NO: 1

DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWIWIRQFPGNKVEWMGYITSSGSTSYNPSLKSRISFTRDTSKNQFFLQLTSVTTEDTATYYCARLDFGNYGGAMDYWGQGTSVTVSS

B3/29F6 VL SEQ ID NO:2

DIVMTQSHKFMSTSIGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYITPYTFGGGTKLEIK

The underlined regions are CDRs (according to the IMGT definition, the sequences are as follows):

Table 2. CDR sequences of mouse anti-TIGIT antibodies

2.2 Construction of IgG1 wild-type chimeric expression vector

Wild-type IgG1 chimeric antibodies and ADCC knockout chimeric antibodies share the same light chain vector. The difference lies in the heavy chain _CH2 amino acids 234 and 235 on the vector containing the human heavy chain constant region sequence: A234/A235 for the ADCC knockout type and L234/L235 for the wild-type type. The method for constructing an IgG1 wild-type chimeric heavy chain expression vector follows the same principles as the expression vector construction method described in 2.1. The heavy chain variable region gene is amplified by PCR using primers containing a leader sequence and then cloned into a vector containing the human heavy chain constant region sequence (CH2 L234/L235) using a seamless ligation method to complete the construction of the IgG1 wild-type chimeric expression vector.

2.3 Expression of chimeric antibodies

The expression vectors obtained in 2.1 and 2.2 were amplified in E. coli and prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) to produce sufficient plasmid for transient transfection and expression of chimeric antibodies. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, #R80007). The two heavy chain vectors and the light chain vector were mixed with polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes, which were then transfected into CHO-S cells and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain an ADCC KO human-mouse chimeric antibody (protein number 900424) and an IgG1 wild-type human-mouse chimeric antibody (protein number 900445).

The chimeric antibody VH and VH sequences obtained according to the above method are as follows:

900423 and 900428 VH SEQ ID NO: 32

DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNRLEWMGYISYSGAPRYNPSLKSRISITRDTSKNQFFLQLSFVTTEDTATYYCASLGTDYYAMDYWGQGTSVIVSS

900424VH SEQ ID NO: 1

DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWIWIRQFPGNKVEWMGYITSSGSTSYNPSLKSRISFTRDTSKNQFFLQLTSVTTEDTATYYCARLDFGNYGGAMDYWGQGTSVTVSS

900424VL SEQ ID NO:2

DIVMTQSHKFMSTSIGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYITPYTFGGGTKLEIK

900423VL SEQ ID NO:34

DIVMTQSHKFMFTSVGDRVSITCKASQHVSNAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFIRGGSGTDFTFTISSVQAEDLAVYYCQQYYSLPWTFGGGTKLEIK

900428VL SEQ ID NO:33

DIVMTQSHKFMFTSVGDRVSITCKASQHVSNAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGRGSGTTDFTFTINSVQAEDLAVYYCQQYYSLPWTFGGGTKLEIK

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 κ light chain constant regions (SEQ ID NO: 31).

2.4 Humanization of mouse anti-human TIGIT antibodies - Preparation of humanized antibodies

The humanization of antibodies was performed by the following method: The variable region sequences of the antibodies were compared with the available sequences in the NCBI IgBlast database, and humanized framework regions (FR regions) suitable for constructing CDR-grafted heavy and light chains were finally determined through identification and analysis.

During the modification, the modification sites were designed based on the conserved amino acid residues in the FR region of human antibodies and the important amino acid residues in the FR region of the antibody. The variable regions of the heavy and light chains of the chimeric antibody were designed for humanized mutations, and PCR technology was used to amplify and construct humanized point mutation antibody expression plasmids. The humanized point mutation antibody expression plasmids were expressed in CHO-S cells and purified to obtain humanized antibody proteins. Using ELISA, Biacore and flow cytometry and other detection methods, the humanized antibodies were screened for indicators such as receptor binding ability, functional inhibitory activity and ADCC effect, and four humanized anti-TIGIT antibodies with excellent performance were obtained. The VH and VL sequences of the obtained humanized anti-TIGIT antibodies are shown below:

900461 and 900464 VH SEQ ID NO: 9

QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGKGLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS

900461VL SEQ ID NO:10

DIQMTQSPSSSLSASVGDRVTITCRASQHVSTAVNWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK

900464 and 900476 VL SEQ ID NO: 11

DIQMTQSPSSSLSASVGDRVTITCKASQHVSTALAWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK

900466VH SEQ ID NO:12

QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGKKLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS

900466VL SEQ ID NO:13

DIQMTQSPSSSLSASVGDRVTITCKASQHVSTAVNWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK

900476VH SEQ ID NO:14

QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGNKLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS

Among them, the underlined region is CDRs (according to the IMGT definition), 900461, 900464, 900466 and 900476 are the numbers of the four humanized antibody proteins, and the corresponding project numbers are HB0030, HB0031, HB0032 and HB0033.

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

Example 3 Detection of chimeric antibodies and humanized antibodies

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

Binding activity detection of chimeric antibodies 900324, 900423, 900424, 900428, and humanized antibodies HB0030, HB0031, HB0032, and HB0033 to cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Huabo Biopharmaceuticals).

All chimeric and humanized antibodies were diluted to 20 μg/ml in PBS containing 1% BSA (1% BSA/PBS), and 20 μL was added to each well of a 96-well U-shaped plate. A negative control (1% BSA/PBS alone) was also established. A suspension of cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Huabo Biotechnology) in the logarithmic growth phase was centrifuged (1000 rpm for 5 minutes), the culture medium discarded, and the cells were resuspended in 1% BSA/PBS to a viable cell density of 1×10 ⁶ /mL. 20 μL (2×10 ⁴ cells) were added to each well of a 96-well U-shaped plate containing anti-TIGIT antibodies and incubated at room temperature for 30 minutes. After the reaction, the 96-well U-shaped plate was resuspended in 1% BSA/PBS, centrifuged (300g×3min), and the supernatant was discarded. The plate was washed once, and 1:200 diluted PE-goat anti-human-Fc (Jackson ImmunoResearch, #109-115-098) was added. The plate was reacted at room temperature in the dark for 15 minutes. After the reaction, the 96-well U-shaped plate was resuspended in 1% BSA/PBS, centrifuged (300g×3min), and the supernatant was discarded. The plate was washed three times, and finally resuspended with 100 μL of 1% BSA/PBS per well. The fluorescence intensity of the PE channel was detected using a flow cytometer (BD, #CantoⅡ).

The binding activity results for the chimeric antibody are shown in Figure 1 and Table 3, and the binding activity results for the humanized antibody are shown in Figure 2 and Table 4. 900324 is Tiragolumab, an anti-human TIGIT antibody from Genentech, transiently expressed in CHO cells. Its light and heavy chain variable region sequences are identical to those of antibody 10A7 in patent application WO2015009856A2. The results showed that both the chimeric and humanized antibodies had good and comparable affinity for cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Huabo Biopharmaceuticals).

Table 3. Binding activity of anti-TIGIT chimeric antibodies to human TIGIT

Antibody 900428 900424 900423 900324 0.075 0.053 0.06 0.124

Table 4. Binding activity of anti-TIGIT humanized antibodies to human TIGIT

Antibody 900324 HB0030 HB0031 HB0032 HB0033 0.08994 0.04661 0.05806 0.07847 0.0514

3.2 Experimental test of TIGIT antibody blocking the binding between TIGIT antigen and TIGIT molecule (competitive method)

The antigen huCD155-moFc (ACROBiosystems, #CD5-H5254) was diluted to 40 μg/ml in PBS containing 1% BSA (1% BSA/PBS). 10 μL was added to each well of a 96-well U-shaped plate and mixed with serially diluted anti-TIGIT antibodies at a 1:1 volume ratio. A positive control (CD155-moFc alone) was also established. A suspension of cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Huabo Bio) in logarithmic growth phase was centrifuged (1000 rpm for 5 min), the culture medium discarded, and the cells resuspended in 1% BSA/PBS to a viable cell density of 1×10 ⁶ /mL. 20 μL (2×10 ⁴ cells) was added to each well of the 96-well U-shaped plate containing the CD155-moFc and anti-TIGIT antibody mixture and incubated at room temperature for 30 min. After the reaction, the 96-well U-shaped plate was resuspended with 1% BSA/PBS, centrifuged (300g×3min), and the supernatant was discarded. The plate was washed once, and 1:300 diluted Alexa488-goat anti-mouse-Fc (Jackson ImmunoResearch, #115-545-071) was added. The plate was reacted at room temperature in the dark for 15 minutes. After the reaction, the 96-well U-shaped plate was resuspended with 1% BSA/PBS, centrifuged (300g×3min), and the supernatant was discarded. The plate was washed three times, and finally resuspended with 100 μL of 1% BSA/PBS per well. The fluorescence intensity of the FITC channel was detected using a flow cytometer (BD, #CantoⅡ).

The results of chimeric antibody binding activity are shown in Figure 3 and Table 5, and the results of humanized antibody binding activity are shown in Figure 4 and Table 6. The results show that chimeric and humanized antibodies have significant blocking effects on human CD155, and the blocking potency is comparable.

Table 5. Blocking activity of anti-TIGIT chimeric antibodies on the binding of human TIGIT to its ligand CD155

Antibody 900428 900424 900423 900324 0.069 0.062 0.065 0.174

Table 6. Blocking activity of anti-TIGIT humanized antibodies on the binding of human TIGIT to its ligand CD155

Antibody 900324 HB0030 HB0031 HB0032 HB0033 0.1349 0.05733 0.06653 0.0919 0.05598

3.3 Affinity testing of humanized monoclonal antibodies

This experiment used the SPR method to determine antigen-antibody binding kinetics and affinity. A BIOCORE (GE, #Biacore8K) was used. A Sereis S Sensor Chip Protein A chip (GE, #29-1275-56) was equilibrated at room temperature for 20–30 min and then loaded into the instrument. Antibody samples were diluted to the experimental working concentration in equilibration buffer and sealed at 2–8°C until ready for use. Antigen and histidine-tagged human TIGIT protein (huTIGIT, ACRO Biosystems, #TIT-H52H3) were diluted in equilibration buffer HBS-EP (10x) (GE, #BR-1006-69). Five concentration gradients of 2.5-fold dilutions were performed, starting at 20 nM, with two replicates of zero concentration (i.e., equilibration buffer) and the lowest concentration. The chip was regenerated with pH 1.5 glycine solution (GE, #BR100354). The samples were analyzed using a capture multi-cycle kinetics program. The corresponding analysis program was selected for data analysis to confirm that there was no obvious reference binding. Kinetics, 1:1 binding model, and fitting analysis were selected to obtain the kinetic parameters of the samples.

The test results are shown in Table 7. The affinity constant (KD(M)) results for huTIGIT showed that the affinity of the humanized monoclonal antibody HB0030 of the present application was close to the order of 10 ^-11 , and had extremely strong affinity.

Table 7. Affinity test results of humanized Mab-TIGIT antibody and hu TIGIT

Antibody Ka(1/Ms) Kd(1/s) KD(M) HB0030 3.17E+06 1.85E-04 5.83E-11

Example 4 Animal in vivo efficacy test

This study investigated the efficacy of anti-human TIGIT antibodies HB0030, HB0031, HB0032, and HB0033 in a CT26.WT colon cancer tumor model in humanized BALB/c-hPD1/hTIGIT mice.

In this experiment, mouse colon cancer cells (CT26.WT) were collected during the logarithmic growth phase, the culture medium was removed, and the cells were washed twice with PBS before inoculation (the viability of CT26.WT cells before and after inoculation was 98.57% and 98.28%, respectively). The inoculum size was 5× ^10⁵ /100 μL per mouse. On day 11 after inoculation, when the average tumor volume reached 94.02 ^mm³ , mice were randomly divided into groups based on tumor volume, with 8 mice per group. Grouping was defined as day 0, and drug administration was performed on days 0, 3, 7, 10, 14, and 17. Following the start of drug administration, tumor size was measured and mice were weighed on days 0, 2, 4, 6, 8, 10, 13, 15, 17, 20, and 23. Tumor volume was calculated as follows: tumor volume ( ^mm³ ) = 0.5 × (longest diameter of tumor × shortest diameter of ^tumor² ). The results are shown in Figure 5.

From the experimental results in FIG5 , it can be seen that the anti-TIGIT humanized monoclonal antibodies HB0030, HB0031, HB0032, and HB0033 of the present application have significant tumor inhibitory effects when administered alone at 10 mg/kg.

The efficacy of HB0030 and the control antibody Tiragolumab at a dose of 10 mg/kg was further studied in the CT26.WT colon cancer tumor model of humanized immune checkpoint mice BALB/c-hPD1/hTIGIT subcutaneously inoculated.

In this experiment, mouse colon cancer cells (CT26) were harvested during the logarithmic growth phase, culture medium removed, and washed twice with PBS before inoculation (CT26 cell survival rates before and after inoculation were 98.51% and 97.250%, respectively). The inoculum size was 5× ^10⁵ /100 μL per mouse. On day 12 of inoculation, when the average tumor volume reached 80.33 ^mm³ , mice were randomly divided into groups based on tumor volume, with 6 mice per group. Grouping was defined as day 0, and drug administration was performed on days 0, 3, 7, 10, 14, and 17. Following the start of drug administration, tumor size was observed and mice were weighed on days 0, 2, 4, 6, 8, 10, 13, 15, 17, 20, and 23. Tumor volume was calculated as follows: tumor volume ( ^mm³ ) = 0.5 × (longest diameter of tumor × shortest diameter of ^tumor² ). The results are shown in Figure 6.

As can be seen from the results in Figure 6, the anti-TIGIT antibody HB0030 of the present application exhibits better tumor inhibition effect compared with the control antibody Tiragolumab.

In this study, there was no significant change in the body weight of mice in all groups 24 days after inoculation.

Sequence Listing

<110> Huabo Biopharmaceutical Technology (Shanghai) Co., Ltd.; Shanghai Huaotai Biopharmaceutical Co., Ltd.

<120> Anti-TIGIT antibodies, preparation methods and applications thereof

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<220>

<223> LCDR3

<400> 8

Gln Gln His Tyr Ile Thr Pro Tyr Thr

1 5

<210> 9

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0031 VH

<400> 9

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ile Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Thr Ser Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Phe Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Phe Gly Asn Tyr Gly Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 VL

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Val Ser Thr Ala

20 25 30

Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 11

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0031 and HB0033 VL

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Ser Thr Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0032 VH

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ile Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Thr Ser Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Phe Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Phe Gly Asn Tyr Gly Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 13

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0032 VL

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln His Val Ser Thr Ala

20 25 30

Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0033 VH

<400> 14

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ile Trp Ile Arg Gln Pro Pro Gly Asn Lys Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Thr Ser Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Phe Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Phe Gly Asn Tyr Gly Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 L-FR1

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

20 25

<210> 16

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0031, HB0032 and HB0033 L-FR1

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 17

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0032 L-FR2

<400> 17

Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 18

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0031 and HB0033 L-FR2

<400> 18

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 19

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 L-FR3

<400> 19

Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 20

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0031 and HB0033 L-FR3

<400> 20

Tyr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 21

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0032 L-FR3

<400> 21

Tyr Arg Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 22

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032 and HB0033 L-FR4

<400> 22

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 23

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032 and HB0033 H-FR1

<400> 23

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser

20 25

<210> 24

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0031 H-FR2

<400> 24

Trp Ile Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

1 5 10 15

Tyr

<210> 25

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0032 H-FR2

<400> 25

Trp Ile Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp Ile Gly

1 5 10 15

Tyr

<210> 26

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0033 H-FR2

<400> 26

Trp Ile Trp Ile Arg Gln Pro Pro Gly Asn Lys Leu Glu Trp Ile Gly

1 5 10 15

Tyr

<210> 27

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032 and HB0033 H-FR3

<400> 27

Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Phe Ser Val Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

20 25 30

Thr Ala Thr Tyr Tyr Cys

35

<210> 28

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032 and HB0033 H-FR4

<400> 28

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 29

<211> 329

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG1 constant region wild type

<400> 29

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 30

<211> 329

<212> PRT

<213> Artificial Sequence

<220>

<223> Human IgG1 constant region mutation

<400> 30

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 31

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Human kappa light chain constant region

<400> 31

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 32

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428VH

<400> 32

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Arg Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ala Pro Arg Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Ser Phe Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Leu Gly Thr Asp Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Ile Val Ser Ser

115

<210> 33

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> 900428VL

<400> 33

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Phe Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln His Val Ser Asn Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

His Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Asn Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423VL

<400> 34

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Phe Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln His Val Ser Asn Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

His Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 35

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423, 900424 and 900428 H-FR1

<400> 35

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr

20 25

<210> 36

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 H-FR2

<400> 36

Trp Ile Trp Ile Arg Gln Phe Pro Gly Asn Lys Val Glu Trp Met Gly

1 5 10 15

Tyr

<210> 37

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 H-FR2

<400> 37

Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Arg Leu Glu Trp Met Gly

1 5 10 15

Tyr

<210> 38

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 H-FR3

<400> 38

Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Phe Thr Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Phe Leu Gln Leu Thr Ser Val Thr Thr Glu Asp

20 25 30

Thr Ala Thr Tyr Tyr Cys

35

<210> 39

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 H-FR3

<400> 39

Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Phe Leu Gln Leu Ser Phe Val Thr Thr Glu Asp

20 25 30

Thr Ala Thr Tyr Tyr Cys

35

<210> 40

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 H-FR4

<400> 40

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 10

<210> 41

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 H-FR4

<400> 41

Trp Gly Gln Gly Thr Ser Val Ile Val Ser Ser

1 5 10

<210> 42

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 HCDR2

<400> 42

Ile Ser Tyr Ser Gly Ala Pro

1 5

<210> 43

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 HCDR3

<400> 43

Ala Ser Leu Gly Thr Asp Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 44

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 L-FR1

<400> 44

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Ile Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser

20 25

<210> 45

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 L-FR1

<400> 45

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Phe Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser

20 25

<210> 46

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 L-FR2

<400> 46

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 47

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 L-FR2

<400> 47

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

1 5 10 15

His

<210> 48

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> 900428 L-FR3

<400> 48

Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Arg Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Phe Thr Ile Asn Ser Val Gln Ala Glu Asp Leu Ala

20 25 30

Val Tyr Tyr Cys

35

<210> 49

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 L-FR3

<400> 49

Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Arg Ser Gly

1 5 10 15

Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala

20 25 30

Val Tyr Tyr Cys

35

<210> 50

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 L-FR3

<400> 50

Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly Arg Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala

20 25 30

Val Tyr Tyr Cys

35

<210> 51

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423, 900424 and 900428 L-FR4

<400> 51

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 52

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 LCDR1

<400> 52

Gln His Val Ser Asn Ala

1 5

<210> 53

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 LCDR3

<400> 53

Gln Gln Tyr Tyr Ser Leu Pro Trp Thr

1 5

<210> 54

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0031 VH; HB0032 VH; HB0033 VH

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa = Lys or Asn

<220>

<221> misc_feature

<222> (45)..(45)

<223> Xaa = Gly or Lys

<400> 54

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ile Trp Ile Arg Gln Pro Pro Gly Xaa Xaa Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Thr Ser Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Phe Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Phe Gly Asn Tyr Gly Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 55

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 VH; HB0030 and HB0031 VH; HB0032 VH; HB0033 VH; 900423 and 900428 VH

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa = Asp or Gln

<220>

<221> misc_feature

<222> (16)..(16)

<223> Xaa = Glu or Gln

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (25)..(25)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (36)..(36)

<223> Xaa = Ile or Asn

<220>

<221> misc_feature

<222> (41)..(41)

<223> Xaa = Phe or Pro

<220>

<221> misc_feature

<222> (44)..(44)

<223> Xaa = Lys or Asn

<220>

<221> misc_feature

<222> (45)..(45)

<223> Xaa = Gly, Lys, or Arg

<220>

<221> misc_feature

<222> (46)..(46)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (49)..(49)

<223> Xaa = Ile or Met

<220>

<221> misc_feature

<222> (53)..(53)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (54)..(54)

<223> Xaa = Ser or Tyr

<220>

<221> misc_feature

<222> (57)..(57)

<223> Xaa = Ala or Ser

<220>

<221> misc_feature

<222> (58)..(58)

<223> Xaa = Pro or Thr

<220>

<221> misc_feature

<222> (59)..(59)

<223> Xaa = Arg, Ser, or Tyr

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa = Ile or Val

<220>

<221> misc_feature

<222> (69)..(69)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (70)..(70)

<223> Xaa = Phe or Ile

<220>

<221> misc_feature

<222> (71)..(71)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (72)..(72)

<223> Xaa = Arg or Val

<220>

<221> misc_feature

<222> (80)..(80)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (82)..(82)

<223> Xaa = Lys or Gln

<220>

<221> misc_feature

<222> (84)..(84)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (85)..(85)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (88)..(88)

<223> Xaa = Ala or Thr

<220>

<221> misc_feature

<222> (89)..(89)

<223> Xaa = Ala or Glu

<220>

<221> misc_feature

<222> (98)..(98)

<223> Xaa = Arg or Ser

<220>

<221> misc_feature

<222> (100)..(100)

<223> Xaa = Asp or Gly

<220>

<221> misc_feature

<222> (101)..(101)

<223> Xaa = Phe or Thr

<220>

<221> misc_feature

<222> (102)..(102)

<223> Xaa = Asp or Gly

<220>

<221> misc_feature

<222> (103)..(103)

<223> Xaa = Asn or Tyr

<220>

<221> misc_feature

<222> (105)..(105)

<223> Xaa = Gly or absent

<220>

<221> misc_feature

<222> (106)..(106)

<223> Xaa = Gly or absent

<220>

<221> misc_feature

<222> (116)..(116)

<223> Xaa = Leu or Ser

<220>

<221> misc_feature

<222> (118)..(118)

<223> Xaa = Ile or Thr

<400> 55

Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Xaa

1 5 10 15

Xaa Leu Ser Leu Thr Cys Thr Val Xaa Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Xaa Trp Ile Arg Gln Xaa Pro Gly Xaa Xaa Xaa Glu Trp

35 40 45

Xaa Gly Tyr Ile Xaa Xaa Ser Gly Xaa Xaa Xaa Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Xaa Xaa Xaa Xaa Xaa Asp Thr Ser Lys Asn Gln Phe Xaa

65 70 75 80

Leu Xaa Leu Xaa Xaa Val Thr Xaa Xaa Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Xaa Leu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Xaa Val Xaa Val Ser Ser

115 120

<210> 56

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424, HB0030, HB0031, HB0032 and HB0033 HCDR2; 900423 and 900428 HCDR2

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = Ser or Tyr

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa = Ala or Ser

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa = Pro or Thr

<400> 56

Ile Xaa Xaa Ser Gly Xaa Xaa

1 5

<210> 57

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423 and 900428 HCDR3; 900424, HB0030, HB0031, HB0032, and HB0033 HCDR3

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Arg or Ser

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa = Asp or Gly

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa = Phe or Thr

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa = Asp or Gly

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa = Asn or Tyr

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa = Gly or absent

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Gly or absent

<400> 57

Ala Xaa Leu Xaa Xaa Xaa Xaa Tyr Xaa Xaa Ala Met Asp Tyr

1 5 10

<210> 58

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032, and HB0033 H-FR1; 900423, 900424, and 900428 H-FR1

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa = Asp or Gln

<220>

<221> misc_feature

<222> (16)..(16)

<223> Xaa = Glu or Gln

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (25)..(25)

<223> Xaa = Ser or Thr

<400> 58

Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Xaa

1 5 10 15

Xaa Leu Ser Leu Thr Cys Thr Val Xaa

20 25

<210> 59

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0031 H-FR2; HB0032 H-FR2; HB0033 H-FR2

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Lys or Asn

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Gly or Lys

<400> 59

Trp Ile Trp Ile Arg Gln Pro Pro Gly Xaa Xaa Leu Glu Trp Ile Gly

1 5 10 15

Tyr

<210> 60

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0031 H-FR2; HB0032 H-FR2; HB0033 H-FR2;

900424 H-FR2; 900423 and 900428 H-FR2

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Ile or Asn

<220>

<221> misc_feature

<222> (7)..(7)

<223> Xaa = Phe or Pro

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Lys or Asn

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Gly, Lys, or Arg

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa = Ile or Met

<400> 60

Trp Xaa Trp Ile Arg Gln Xaa Pro Gly Xaa Xaa Xaa Glu Trp Xaa Gly

1 5 10 15

Tyr

<210> 61

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032 and HB0033 H-FR3; 900423 and 900428 H-FR3;

900424 H-FR3

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa = Arg, Ser, or Tyr

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Ile or Val

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa = Phe or Ile

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (14)..(14)

<223> Xaa = Arg or Val

<220>

<221> misc_feature

<222> (22)..(22)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa = Lys or Gln

<220>

<221> misc_feature

<222> (26)..(26)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (27)..(27)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (30)..(30)

<223> Xaa = Ala or Thr

<220>

<221> misc_feature

<222> (31)..(31)

<223> Xaa = Ala or Glu

<400> 61

Xaa Tyr Asn Pro Ser Leu Lys Ser Arg Xaa Xaa Xaa Xaa Xaa Asp Thr

1 5 10 15

Ser Lys Asn Gln Phe Xaa Leu Xaa Leu Xaa Xaa Val Thr Xaa Xaa Asp

20 25 30

Thr Ala Thr Tyr Tyr Cys

35

<210> 62

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030, HB0031, HB0032 and HB0033 H-FR4; 900424 H-FR4;

900423 and 900428 H-FR4

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa = Leu or Ser

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa = Ile or Thr

<400> 62

Trp Gly Gln Gly Thr Xaa Val Xaa Val Ser Ser

1 5 10

<210> 63

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 VL; HB0031 and HB0033 VL; HB0032 VL

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa = Lys or Arg

<220>

<221> misc_feature

<222> (33)..(33)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa = Ala or Asn

<220>

<221> misc_feature

<222> (54)..(54)

<223> Xaa = Leu or Arg

<220>

<221> misc_feature

<222> (55)..(55)

<223> Xaa = Gln or Tyr

<220>

<221> misc_feature

<222> (66)..(66)

<223> Xaa = Gly or Arg

<400> 63

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Xaa Ala Ser Gln His Val Ser Thr Ala

20 25 30

Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Xaa Xaa Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Xaa Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 64

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424 VL; HB0030 VL; HB0031 and HB0033 VL; HB0032 VL;

900423VL; 900428VL

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = Gln or Val

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa = His or Pro

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa = Lys or Ser

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Leu or Met

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa = Ala or Thr

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa = Ile or Val

<220>

<221> misc_feature

<222> (20)..(20)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa = Lys or Arg

<220>

<221> misc_feature

<222> (31)..(31)

<223> Xaa = Asn or Thr

<220>

<221> misc_feature

<222> (33)..(33)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (34)..(34)

<223> Xaa = Ala or Asn

<220>

<221> misc_feature

<222> (42)..(42)

<223> Xaa = Lys or Gln

<220>

<221> misc_feature

<222> (43)..(43)

<223> Xaa = Ala or Ser

<220>

<221> misc_feature

<222> (49)..(49)

<223> Xaa = His or Tyr

<220>

<221> misc_feature

<222> (54)..(54)

<223> Xaa = Leu or Arg

<220>

<221> misc_feature

<222> (55)..(55)

<223> Xaa = Gln or Tyr

<220>

<221> misc_feature

<222> (56)..(56)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (60)..(60)

<223> Xaa = Asp or Ser

<220>

<221> misc_feature

<222> (63)..(63)

<223> Xaa = Ile, Ser, or Thr

<220>

<221> misc_feature

<222> (65)..(65)

<223> Xaa = Arg or Ser

<220>

<221> misc_feature

<222> (66)..(66)

<223> Xaa = Gly or Arg

<220>

<221> misc_feature

<222> (73)..(73)

<223> Xaa = Phe or Leu

<220>

<221> misc_feature

<222> (76)..(76)

<223> Xaa = Asn or Ser

<220>

<221> misc_feature

<222> (78)..(78)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (80)..(80)

<223> Xaa = Ala or Pro

<220>

<221> misc_feature

<222> (83)..(83)

<223> Xaa = Phe or Leu

<220>

<221> misc_feature

<222> (85)..(85)

<223> Xaa = Thr or Val

<220>

<221> misc_feature

<222> (91)..(91)

<223> Xaa = His or Tyr

<220>

<221> misc_feature

<222> (93)..(93)

<223> Xaa = Ile or Ser

<220>

<221> misc_feature

<222> (94)..(94)

<223> Xaa = Leu or Thr

<220>

<221> misc_feature

<222> (96)..(96)

<223> Xaa = Trp or Tyr

<220>

<221> misc_feature

<222> (100)..(100)

<223> Xaa = Gly or Gln

<400> 64

Asp Ile Xaa Met Thr Gln Ser Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Gly

1 5 10 15

Asp Arg Val Xaa Ile Thr Cys Xaa Ala Ser Gln His Val Ser Xaa Ala

20 25 30

Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly Xaa Xaa Pro Lys Leu Leu Ile

35 40 45

Xaa Ser Ala Ser Tyr Xaa Xaa Xaa Gly Val Pro Xaa Arg Phe Xaa Gly

50 55 60

Xaa Xaa Ser Gly Thr Asp Phe Thr Xaa Thr Ile Xaa Ser Xaa Gln Xaa

65 70 75 80

Glu Asp Xaa Ala Xaa Tyr Tyr Cys Gln Gln Xaa Tyr Xaa Xaa Pro Xaa

85 90 95

Thr Phe Gly Xaa Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 65

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424, HB0030, HB0031, HB0032, and HB0033 LCDR1; 900423 and 900428 LCDR1

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa = Asn or Thr

<400> 65

Gln His Val Ser Xaa Ala

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 900424, HB0030, HB0031, HB0032, and HB0033 LCDR3; 900423 and 900428 LCDR3

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = His or Tyr

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa = Ile or Ser

<220>

<221> misc_feature

<222> (6)..(6)

<223> Xaa = Leu or Thr

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa = Trp or Tyr

<400> 66

Gln Gln Xaa Tyr Xaa Xaa Pro Xaa Thr

1 5

<210> 67

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0031, HB0032, and HB0033 L-FR1; HB0030 L-FR1

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa = Lys or Arg

<400> 67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Xaa Ala Ser

20 25

<210> 68

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 L-FR1; HB0031, HB0032, and HB0033 L-FR1;

900424 L-FR1; 900423 and 900428 L-FR1

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = Gln or Val

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa = His or Pro

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa = Lys or Ser

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Leu or Met

<220>

<221> misc_feature

<222> (12)..(12)

<223> Xaa = Phe or Ser

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa = Ala or Thr

<220>

<221> misc_feature

<222> (15)..(15)

<223> Xaa = Ile or Val

<220>

<221> misc_feature

<222> (20)..(20)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa = Lys or Arg

<400> 68

Asp Ile Xaa Met Thr Gln Ser Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Gly

1 5 10 15

Asp Arg Val Xaa Ile Thr Cys Xaa Ala Ser

20 25

<210> 69

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0032 L-FR2; HB0031 and HB0033 L-FR2

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Ala or Asn

<400> 69

Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

1 5 10 15

Tyr

<210> 70

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 and HB0032 L-FR2; HB0031 and HB0033 L-FR2;

900424 L-FR2; 900423 and 900428 L-FR2

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Ala or Asn

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa = Lys or Gln

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Ala or Ser

<220>

<221> misc_feature

<222> (17)..(17)

<223> Xaa = His or Tyr

<400> 70

Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly Xaa Xaa Pro Lys Leu Leu Ile

1 5 10 15

Xaa

<210> 71

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 L-FR3; HB0031 and HB0033 L-FR3; HB0032 L-FR3

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Leu or Arg

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = Gln or Tyr

<220>

<221> misc_feature

<222> (14)..(14)

<223> Xaa = Gly or Arg

<400> 71

Tyr Xaa Xaa Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Xaa Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 72

<211> 36

<212> PRT

<213> Artificial Sequence

<220>

<223> HB0030 L-FR3; HB0031 and HB0033 L-FR3; HB0032 L-FR3;

900428 L-FR3; 900424 L-FR3; 900423 L-FR3

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa = Leu or Arg

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = Gln or Tyr

<220>

<221> misc_feature

<222> (4)..(4)

<223> Xaa = Ser or Thr

<220>

<221> misc_feature

<222> (8)..(8)

<223> Xaa = Asp or Ser

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa = Ile, Ser, or Thr

<220>

<221> misc_feature

<222> (13)..(13)

<223> Xaa = Arg or Ser

<220>

<221> misc_feature

<222> (14)..(14)

<223> Xaa = Gly or Arg

<220>

<221> misc_feature

<222> (21)..(21)

<223> Xaa = Phe or Leu

<220>

<221> misc_feature

<222> (24)..(24)

<223> Xaa = Asn or Ser

<220>

<221> misc_feature

<222> (26)..(26)

<223> Xaa = Leu or Val

<220>

<221> misc_feature

<222> (28)..(28)

<223> Xaa = Ala or Pro

<220>

<221> misc_feature

<222> (31)..(31)

<223> Xaa = Phe or Leu

<220>

<221> misc_feature

<222> (33)..(33)

<223> Xaa = Thr or Val

<400> 72

Tyr Xaa Xaa Xaa Gly Val Pro Xaa Arg Phe Xaa Gly Xaa Xaa Ser Gly

1 5 10 15

Thr Asp Phe Thr Xaa Thr Ile Xaa Ser Xaa Gln Xaa Glu Asp Xaa Ala

20 25 30

Xaa Tyr Tyr Cys

35

<210> 73

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> 900423, 900424, and 900428 L-FR4; HB0030, HB0031, HB0032, and HB0033 L-FR4

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa = Gly or Gln

<400> 73

Phe Gly Xaa Gly Thr Lys Leu Glu Ile Lys

1 5 10

Claims (19)

1. An isolated antigen-binding protein comprising a heavy chain variable region VH, wherein the VH comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:5; and comprising a light chain variable region VL, wherein the VL comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:6, the amino acid sequence of LCDR2 is shown in SEQ ID NO:7, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:8. 2. The isolated antigen-binding protein according to claim 1, comprising a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 12 and 14. 3. The isolated antigen-binding protein according to claim 1, comprising a light chain variable region VL, wherein the VL comprises the amino acid sequence shown in any one of SEQ ID NO: 2, 10, 11 and 13. 4. The isolated antigen-binding protein according to claim 1, comprising a heavy chain variable region VH and a light chain variable region VL, and the isolated antigen-binding protein comprising an amino acid sequence selected from any one of the following groups: (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, and (5) VH: SEQ ID NO: 1, VL: SEQ ID NO: 2. 5. The isolated antigen-binding protein according to claim 1, having one or more of the following properties: 1) Capable of binding TIGIT protein with a KD value of 1× ^10⁻¹⁰ M or lower, wherein the KD value is determined by surface plasmon resonance method; 2) In FACS assays, it can block the binding of CD155 to TIGIT; and 3) It can inhibit tumor growth and/or tumor cell proliferation. 6. The isolated antigen-binding protein according to claim 1, comprising an antibody or an antigen-binding fragment thereof. 7. The isolated antigen-binding protein according to claim 6, wherein the antigen-binding fragment comprises Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb. 8. The isolated antigen-binding protein according to claim 6, wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, and humanized antibodies. 9. The isolated antigen-binding protein according to claim 1, comprising an antibody light chain constant region, wherein the antibody light chain constant region comprises a human κ light chain constant region. 10. The isolated antigen-binding protein according to claim 9, wherein the human κ light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 31. 11. The isolated antigen-binding protein according to claim 1, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region comprises a human IgG constant region. 12. The isolated antigen-binding protein according to claim 11, wherein the antibody heavy chain constant region comprises a human IgG1 constant region, and the human IgG1 constant region comprises the amino acid sequence shown in any one of SEQ ID NO:29-30. 13. One or more isolated nucleic acid molecules encoding the isolated antigen-binding protein of any one of claims 1-12. 14. A vector comprising the nucleic acid molecule according to claim 13. 15. A cell comprising a nucleic acid molecule according to claim 13 or a vector according to claim 14. 16. A method for preparing the isolated antigen-binding protein of any one of claims 1-12, the method comprising culturing the cells of claim 15 under conditions that cause expression of the isolated antigen-binding protein of any one of claims 1-12. 17. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-12, the nucleic acid molecule of claim 13, the carrier of claim 14, and/or the cell of claim 15, and optionally a pharmaceutically acceptable adjuvant. 18. Use of the isolated antigen-binding protein of any one of claims 1-12, the nucleic acid molecule of claim 13, the carrier of claim 14, the cell of claim 15, and/or the pharmaceutical composition of claim 17 in the preparation of a medicament for the prevention, relief, and/or treatment of TIGIT-related disease, wherein the TIGIT-related disease is colon cancer. 19. Use of the isolated antigen-binding protein of any one of claims 1-12 in the preparation of a reagent for inhibiting the binding of CD155 to TIGIT.