WO2021227940A1 - 抗tigit的抗体、其制备方法和应用 - Google Patents

抗tigit的抗体、其制备方法和应用 Download PDF

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WO2021227940A1
WO2021227940A1 PCT/CN2021/092043 CN2021092043W WO2021227940A1 WO 2021227940 A1 WO2021227940 A1 WO 2021227940A1 CN 2021092043 W CN2021092043 W CN 2021092043W WO 2021227940 A1 WO2021227940 A1 WO 2021227940A1
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seq
antigen binding
binding protein
amino acid
acid sequence
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PCT/CN2021/092043
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English (en)
French (fr)
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于海佳
蔡明清
韦小越
陈时
谢剑梅
朱向阳
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华博生物医药技术（上海）有限公司
上海华奥泰生物药业股份有限公司
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Priority to JP2022567570A priority Critical patent/JP2023534588A/ja
Priority to KR1020227042983A priority patent/KR20230009441A/ko
Priority to CN202180033811.XA priority patent/CN115605511A/zh
Priority to US17/923,999 priority patent/US20230183345A1/en
Priority to EP21804577.1A priority patent/EP4148068A1/en
Priority to AU2021273240A priority patent/AU2021273240A1/en
Publication of WO2021227940A1 publication Critical patent/WO2021227940A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of biomedicine, in particular to an antigen binding protein targeting TIGIT, its preparation method and application.
  • T cells Under normal circumstances, T cells rely on exogenous or endogenous antigen-MHC-I/II complexes presented by antigen-presenting cells (APCs) and T cell (CD4 + , CD8 + ) surface receptors ( T Cell Receptor (TCR) is combined with additional costimulatory (Costimulatory) signals to fully activate, and then other effector cells are used to kill cells expressing related antigens.
  • APCs antigen-presenting cells
  • T cell CD4 + , CD8 + ) surface receptors
  • T Cell Receptor T Cell Receptor
  • T cells are also regulated by coinhibitory signals. The latter has a negative regulatory effect on the activity, proliferation, and survival of T cells. It can also maintain self-tolerance, prevent excessive autoimmune reactions, and reduce immunity. Tissue damage caused by response.
  • the activation and inhibition of T cells are in an abnormal mode. At present, their normalization is widely regarded as an important means of tumor treatment.
  • TIGIT T cell Ig and ITIM domain, also known as WUCAM, Vstm3 or VSIG9
  • PVR poliovirus receptor
  • Nectin poliovirus receptor
  • IgV extracellular immunoglobulin variable region
  • ITIM immunoreceptor tyrosine inhibitory motif
  • ITT immunoglobulin tyrosine tail
  • the TIGIT gene is located on human chromosome 16 and encodes a type I transmembrane protein composed of 244 amino acids.
  • the extracellular region of human TIGIT molecule is 141 amino acids long and has 1 immunoglobulin V-like domain; the transmembrane region has 23 amino acids; the cytoplasmic region is shorter, with 80 amino acids, and has 1 PDZ binding domain and 1 An ITIM phantom.
  • CD155 (also called PVR, Necl5 or Tage4) is a high-affinity ligand of TIGIT. Once CD155, which is highly expressed on the tumor surface, binds to the 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; inhibiting the co-stimulatory ability of dendritic cells, leading to a decrease in cancer antigen presentation and an increase in anti-inflammatory cytokines such as IL-10.
  • TIGIT can also induce other cells such as PVR signal transduction of tumor cells; TIGIT can directly inhibit CD8 + T cell effects, or TIGIT + Treg can inhibit CD8 + T cells and prevent the clearance of cancer cells (Manieri et al., Inhibition of the Cancer Immunity Cycle by TIGIT.2016) .
  • TIGIT will become a suitable target for patients with cancer and other diseases. Therefore, in view of the role and function of TIGIT in various related diseases, there is still a need to develop improved anti-TIGIT specificity suitable for the treatment of patients in this field. Antibody.
  • the present application provides an isolated antigen binding protein that can include at least one CDR in the VH of the heavy chain variable region, and the VH includes the amino acid sequence shown in SEQ ID NO:55.
  • the isolated antigen binding protein may also include at least one CDR in the light chain variable region VL, and the VL includes the amino acid sequence shown in SEQ ID NO:64.
  • the isolated antigen binding protein has one or more of the following properties: 1) It can be 1 ⁇ 10 -10 M or less (for example, 8 ⁇ 10 -11 M, 5 ⁇ 10 -11 M, 2 ⁇ 10 -11 M, 1 ⁇ 10 -11 M or lower) KD value combined with TIGIT protein, wherein the KD value is measured by surface plasmon resonance method; 2) In FACS measurement, it can block CD155 and TIGIT Combined; and 3) capable of inhibiting tumor growth and/or tumor cell proliferation.
  • the present application provides an isolated antigen binding protein comprising at least one CDR in the VH of the heavy chain variable region, the VH comprising the amino acid sequence shown in SEQ ID NO: 55.
  • the isolated antigen binding protein comprises at least one CDR in the light chain variable region VL, and 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) It can bind to the TIGIT protein with a KD value of 1 ⁇ 10 -10 M or lower, wherein the KD The value is determined by the surface plasmon resonance method; 2) it can block the binding of CD155 and TIGIT in the FACS measurement; and 3) it can inhibit tumor growth and/or tumor cell proliferation.
  • 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, and the reference antibody
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 56; and the HCDR3 includes SEQ ID NO: 57, the light chain variable region of the reference antibody includes LCDR1, LCDR2 and LCDR3, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 65; the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 7 The LCDR3 includes the amino acid sequence shown in SEQ ID NO: 66.
  • the isolated antigen binding protein includes an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:56.
  • the HCDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 4 and 42.
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 57.
  • the HCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 5 and 43.
  • the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the isolated antigen binding protein comprises any set of amino acid sequences selected from the following:
  • HCDR1 SEQ ID NO: 3
  • HCDR2 SEQ ID NO: 4
  • HCDR3 SEQ ID NO: 5;
  • HCDR1 SEQ ID NO: 3
  • HCDR2 SEQ ID NO: 42
  • HCDR3 SEQ ID NO: 43.
  • the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:65.
  • the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 6 and 52.
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:7.
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 66.
  • the LCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 8 and 53.
  • the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, and the isolated antigen binding protein comprises any set of amino acid sequences selected from the following:
  • LCDR1 SEQ ID NO: 6
  • LCDR2 SEQ ID NO: 7
  • LCDR3 SEQ ID NO: 8;
  • the VL includes the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 68.
  • the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 15, 16, 44, and 45.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 70.
  • the L-FR2 includes the amino acid sequence shown in any one of SEQ ID Nos: 17, 18, 46, and 47.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 72.
  • the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID Nos: 19, 20, 21, 48, 49, and 50.
  • the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 73.
  • the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 22 and 51.
  • the VL includes the amino acid sequence shown in SEQ ID NO:64.
  • the VL comprises the amino acid sequence shown in any one of SEQ ID NO: 2, 10, 11, 13, 33, and 34.
  • the isolated antigen binding protein includes an antibody light chain constant region, and the antibody light chain constant region is derived from a human kappa light chain constant region, and the human kappa light chain constant region comprises SEQ ID The amino acid sequence shown in NO:31.
  • the VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO:58.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 23 and 35 .
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO:60.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the sequence shown in any one of SEQ ID NOs: 24, 25, 26, 36, and 37 Amino acid sequence.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 61.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 27, 38, and 39.
  • the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 62.
  • the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in any one of SEQ ID NOs: 28, 40, and 41.
  • the VH comprises the amino acid sequence shown in SEQ ID NO:55.
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 9, 12, 14 and 32.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.
  • the antibody heavy chain constant region includes a human IgG1 constant region, and the human IgG1 constant region includes the amino acid sequence shown in any one of SEQ ID NO: 29-30.
  • the antigen binding protein comprises a heavy chain variable region VH and a light chain variable region VL
  • the isolated antigen binding protein comprises any set of amino acid sequences selected from the following: (1) VH: SEQ ID NO: 9, VL: SEQ ID NO: 10, (2) VH: SEQ ID NO: 9, VL: SEQ ID NO: 11, (3) VH: SEQ ID NO: 12, VL: SEQ ID NO: 13, (4) VH: SEQ ID NO: 14, VL: SEQ ID NO: 11, (5) VH: SEQ ID NO: 1, VL: SEQ ID NO: 2, (6) VH: SEQ ID NO :32, VL: SEQ ID NO: 34, and (7)
  • VH SEQ ID NO: 32
  • VL SEQ ID NO: 33.
  • the present application provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein.
  • the present application provides a vector, which contains the nucleic acid molecule.
  • the present application provides a cell comprising the nucleic acid molecule or the vector.
  • the present application provides a polypeptide comprising the isolated antigen binding protein.
  • the present application provides an immunoconjugate comprising the isolated antigen binding protein or the polypeptide.
  • the present application provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under the condition that the isolated antigen binding protein is expressed.
  • the present application provides a pharmaceutical composition, which comprises the isolated antigen binding protein, the nucleic acid molecule, the carrier, the polypeptide, the immunoconjugate and/ Or the cell, and optionally a pharmaceutically acceptable adjuvant.
  • the present application provides the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell, the polypeptide, the immunoconjugate and/or the Use of the pharmaceutical composition in the preparation of a medicine for the prevention, alleviation and/or treatment of TIGIT-related diseases.
  • the TIGIT-related disease is a T cell dysfunction disorder.
  • the TIGIT-related disease is tumor.
  • the tumor includes a solid tumor and/or a non-solid tumor.
  • 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.
  • the present application provides a method for inhibiting the binding of CD155 to TIGIT, the method comprising administering the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the The polypeptide, the immunoconjugate and/or the pharmaceutical composition.
  • the present application provides the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell, the polypeptide, the immunoconjugate and/or the A pharmaceutical composition for preventing, alleviating or treating TIGIT-related diseases.
  • the present application provides a method for preventing, alleviating or treating TIGIT-related diseases, the method comprising administering the isolated antigen binding protein, the nucleic acid molecule, the The carrier, the cell, the polypeptide, the immunoconjugate and/or the pharmaceutical composition.
  • the TIGIT-related disease is a T cell dysfunction disorder.
  • the TIGIT-related disease is tumor.
  • the tumor includes a solid tumor and/or a non-solid tumor.
  • 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.
  • Figure 1 shows the binding activity of TIGIT chimeric antibody to cells expressing human TIGIT.
  • Figure 2 shows the binding activity of TIGIT humanized antibodies to cells expressing human TIGIT.
  • Figure 3 shows the blocking activity of the TIGIT chimeric antibody against the binding of human TIGIT to its ligand CD155.
  • Figure 4 shows the blocking activity of TIGIT humanized antibody against the binding of human TIGIT to its ligand CD155.
  • FIG. 5 shows the efficacy test of TIGIT antibody in animals.
  • FIG. 6 shows the comparison of the efficacy of TIGIT antibody with the control antibody Tiragolumab in animals.
  • FIG. 7 shows the results of the efficacy experiment of TIGIT antibody in animals.
  • TIGIT is referred to as "T cell immune receptor containing Ig and ITIM domains", which is usually a member of the PVR (poliovirus receptor) family of immunoglobulins, which can bind to PVR/ CD155 and Nectin-2/CD112.
  • PVR poliovirus receptor
  • the TIGIT in the present application can refer to the TIGIT protein from any vertebrate source, including mammals, such as primates (e.g., humans, rhesus monkeys, and cynomolgus monkeys) and rodents (e.g., mice and rats).
  • TIGIT encompasses 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.
  • TIGIT is human TIGIT, and the amino acid sequence of human TIGIT includes the amino acid sequence shown in UniProt accession number Q495A1.
  • TIGIT is cynomolgus TIGIT, and the amino acid sequence of cynomolgus TIGIT includes the amino acid sequence shown in UniProt accession number G7NXM4.
  • TIGIT is mouse TIGIT, and the amino acid sequence of mouse TIGIT includes the amino acid sequence shown in UniProt accession number P86176.
  • the term "antigen-binding protein” generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugates Compounds, multispecific antibodies (for example, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen-binding fragments
  • Fv fragments fragments
  • F(ab') 2 fragments
  • scFv di-scFv and/or dAb
  • immunoconjugates Compounds, multispecific antibodies (for example, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain
  • F(ab ') 2 generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region.
  • Fv generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment may be composed of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound;
  • dsFv generally refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • dAb fragment generally refers to an antibody fragment composed of a VH domain.
  • scFv generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • an “antibody” is used in the broadest sense, and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , Multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • An “antibody” can generally comprise a protein of at least two heavy chains (HC) and two light chains (LC) connected to each other by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region contains three domains, CH1, CH2, and CH3.
  • each light chain contains a light chain variable region (VL) and a light chain constant region.
  • the constant region of the light chain contains one domain, CL.
  • the VH and VL regions can be further subdivided into hyperdenatured regions, called complementarity determining regions (CDR), which alternate with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL contains three CDRs and four framework regions (FR), which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • variable domains of the natural heavy chain and light chain each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) Most of them adopt the ⁇ -sheet configuration, connected by three CDRs to form a loop connection, and in some cases form part of the ⁇ -sheet structure.
  • the CDRs in each chain are closely brought together by the FR region, and together with the CDRs from the other chain, form the antigen binding site of the antibody.
  • the constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light chain and heavy chain variable regions, which are called complementarity determining regions (CDR) or hypervariable regions (HVR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)), Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997) and concepts based on IMGT-ONTOLOGY (IMGT-ONTOLOGY) IMGT definition rules and IMGT Scientific chart rules.
  • IMGT refers to the International ImmunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http://www.imgt.org). IMGT specializes in immunoglobulin (IG) or antibodies from humans and other vertebrates, T cell receptor (TR), major histocompatibility (MH), and immunoglobulin superfamily from vertebrates and invertebrates (IgSF), MH superfamily (MhSF) and immune system related protein (RPI).
  • IG immunoglobulin
  • TR T cell receptor
  • MH major histocompatibility
  • IgSF immunoglobulin superfamily from vertebrates and invertebrates
  • MhSF MH superfamily
  • RPI immune system related protein
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, separated and/or recovered from a component of its production environment (for example, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic uses, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated antigen binding protein or antibody will generally be prepared through at least one purification step.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies in the group are the same, except for a small number of natural mutations that may exist.
  • Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • the modifier "monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not to be interpreted as requiring the production of the antibody by any specific method.
  • the monoclonal antibodies used in the present application can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.
  • 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.
  • 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 compared with the parental (e.g., mouse-derived) antibody in a human individual The possibility of triggering an adverse immune response is reduced.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be allowed, as long as they still retain the ability of the antibody to bind to a specific antigen.
  • the humanized antibody may optionally comprise at least a portion of the constant region of a human immunoglobulin. "Humanized antibodies” retain antigen specificity similar to the original antibodies.
  • “Humanized” forms of non-human (e.g., murine) antibodies may contain minimally chimeric antibodies derived from non-human immunoglobulin sequences.
  • the CDR region residues in human immunoglobulin (acceptor antibody) can be equipped with non-human species (donor antibody) (such as mouse, rat) with the desired properties, affinity, and/or ability.
  • donor antibody such as mouse, rat
  • Rabbit or non-human primate residues in the CDR region In some cases, the FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.
  • Fully human antibody generally refers to an antibody that is expressed by transferring a human antibody-encoding gene to a genetically engineered antibody gene-deficient animal. 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 on the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
  • binding 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 molecule
  • targets in the context of heterogeneous populations (including biological molecules).
  • 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.
  • an antibody that specifically binds to a target is an antibody that binds to this target with greater affinity, affinity, easier and/or longer duration than it binds to other targets.
  • epitope When an antibody binds to an epitope through its antigen binding domain more easily than it will bind to a random, unrelated epitope, the antibody is said to "specifically bind” to that antigen.
  • epitope refers to a specific atomic group (for example, sugar side chain, phosphoryl, sulfonyl) or amino acid that binds to an antigen-binding protein (such as an antibody) on an antigen.
  • KD and “K D” are used interchangeably, usually referring to the equilibrium dissociation constant
  • KD is the dissociation rate constant (kdis, also known as the “off-rate ) (koff)” or “kd") and the ratio of the association rate constant (kon, also known as “combination rate (kon)” or “ka”).
  • the binding rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD) can be used to express the binding affinity of an antigen-binding protein (for example, an antibody) to an antigen.
  • Methods for determining the rate constants of association and dissociation are well known in the art, including but not limited to Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , Co-immunoprecipitation (Co-IP) and protein chip technology. If measured under different conditions (such as salt concentration, pH), the measured affinity of a specific protein-protein interaction can be different.
  • reference antibody generally refers to an antibody with which the antigen binding protein described in this application competes for binding to the antigen TIGIT.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic effector cells eg, NK cells
  • FcR Fc receptors
  • the main cells that mediate ADCC (such as NK cells) only express Fc ⁇ RIII, while monocytes express Fc ⁇ RI, Fc ⁇ RII and Fe ⁇ RIII (see Ravetch and Kinet, Annu. Rev. Immunol.
  • in vitro and/or in vivo cytotoxicity assays can be performed to evaluate the ADCC activity of the target molecule.
  • in vitro ADCC assays can be performed, see U.S. Patent No. 5,500,362 or No. 5,821,337 or U.S. Patent No. 6,737,056 (Presta) Recorded.
  • Useful effector cells for such assays include PBMC and NK cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).
  • an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fc ⁇ R binding (and therefore may lack ADCC activity), but retains FcRn binding ability.
  • ADCC activity can be reduced by modifying the Fc region.
  • sites that affect binding to Fc receptors can be removed, for example, sites that are not salvage receptor binding sites are removed.
  • the Fc region can be modified to remove ADCC sites.
  • ADCC sites are known in the art. For ADCC sites of IgG1, see, for example, Sarmay et al. (1992) Molec. Immunol. 29(5):633-9.
  • the term "between” usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. Indirect connection.
  • the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1
  • the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2
  • the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3.
  • the "first amino acid fragment" and the "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • the term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, separated and/or recovered from a component of its production environment (for example, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic uses, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated antigen binding protein or antibody will generally be prepared through at least one purification step.
  • the isolated antigen binding proteins described in this application generally do not bind to antigens other than TIGIT antigens.
  • isolated nucleic acid molecule or isolated polynucleotide
  • isolated nucleic acid molecule generally refers to genome, mRNA, cDNA, or synthetic origin DNA or RNA or a certain combination thereof, which is not compatible with the polynucleus found in nature. All or part of the nucleotides are associated or linked to polynucleotides to which they are not linked in nature.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having 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. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the term "cell” generally refers to an individual cell, cell line, or cell line that can or already contains a plasmid or vector containing the nucleic acid molecule described in this application, or capable of expressing the antibody or antigen-binding fragment thereof described in this application.
  • the cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be identical in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application.
  • the cells can be obtained by transfecting cells in vitro using the vectors described in this application.
  • the cell may be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells).
  • the cell may be a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector is introduced.
  • the recombinant host cell includes not only certain specific cells, but also the progeny of these cells.
  • the term "pharmaceutically acceptable adjuvant” generally includes pharmaceutically acceptable carriers, excipients or stabilizers, which are not effective for the cells or mammals exposed to them at the dose and concentration used. Poisonous.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers may include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin Or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, Mannose or dextrin; 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 TM , polyethylene glycol (PEG ) And PLURONICS TM .
  • buffers such as phosphate, citrate and other organic acids
  • antioxidants including ascorbic acid
  • administering refers to the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs, or biological fluids.
  • administering can refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells.
  • administering also mean treatment by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to humans, animals or research subjects, refers to treatment, preventive or preventive measures, research and diagnosis; including TIGIT conjugates and humans or animals, subjects, cells, tissues, physiological regions Chamber or physiological fluid contact.
  • treatment refers to the administration of an internal or external therapeutic agent, including any one of the TIGIT antigen binding proteins and compositions of the present application, to a patient who has one or more disease symptoms, and the patient is known to The therapeutic agent has a therapeutic effect on these symptoms.
  • the patient is administered in an amount (therapeutically effective amount) of a therapeutic agent effective to alleviate the symptoms of one or more diseases.
  • the desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis.
  • one or more symptoms related to cancer are alleviated or eliminated, including but not limited to, reducing (or destroying) the proliferation of cancer cells, reducing the symptoms derived from the disease, and improving the quality of life of those suffering from the disease .
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all pre-cancerous and cancerous cells and tissues.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an isolated antigen binding protein, which may include at least one CDR in a heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 55.
  • the present application provides an isolated antigen binding protein, which may include at least one CDR in a heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 54.
  • the isolated antigen binding protein may comprise at least one CDR in the VH of the heavy chain variable region, and the VH may comprise any one of SEQ ID NOs: 1, 9, 12, 14 and 32.
  • the amino acid sequence may comprise any one of SEQ ID NOs: 1, 9, 12, 14 and 32.
  • the VH of the antigen binding protein may include HCDR1, HCDR2 and HCDR3.
  • the antigen binding protein described in the present application may include HCDR1, wherein the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3: GYSITSDYA.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise 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, and X 3 is S or Y, X 6 is A or S, and X 7 is P or T.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 4 and 42.
  • the antigen binding protein described in the present application may include HCDR2, wherein the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 4: ITSSGST.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57: AX 2 LX 4 X 5 X 6 X 7 YX 9 X 10 AMDY, wherein X 2 is R or S, X 4 is D or G, X 5 is F or T, X 6 is D or G, X 7 is N or Y, X 9 is G or absent, and X 10 is G or absent.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 5 and 43.
  • the antigen binding protein described in the present application may include HCDR3, wherein the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 5: ARLDFGNYGGAMDY.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may also include framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the antigen binding protein described in the present application may comprise the framework region H-FR1, wherein the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 58: X 1 VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 , where X 1 is D Or Q, X 16 is E or Q, X 17 is S or T, and X 25 is S or T.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 23 and 35.
  • the antigen binding protein described in the present application may include the framework region H-FR1, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 23: QVQLQESGPGLVKPSETLSLTCTVS.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise the framework region H-FR2, wherein the H-FR2 may comprise the amino acid sequence shown 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 24-26 and 36-37.
  • the antigen binding protein described in the present application may comprise the framework region H-FR2, wherein the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 59: WIWIRQPPGX 10 X 11 LEWIGY; wherein X 10 is K or N, X 11 is G or K.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 24-26.
  • the antigen binding protein described in the present application may comprise the framework region H-FR3, wherein the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 61: X 1 YNPSLKSRX 10 X 11 X 12 X 13 X 14 DTSKNQFX 22 LX 24 LX 26 X 27 VTX 30 X 31 DTATYYC, where X 1 is R, S or Y, X 10 is I or V, X 11 is S or T, X 12 is F or I, X 13 is S or T, X 14 is R or V, X 22 is F or S, X 24 is K or Q, X 26 is S or T, X 27 is F or S, X 30 is A or T, and X 31 is A or E .
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 27, 38, and 39.
  • the antigen binding protein described in the present application may include the framework region H-FR3, wherein the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 27: YYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise the 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR4 may include the amino acid sequence shown in any one of SEQ ID NO: 28, 40, and 41.
  • the antigen binding protein described in the present application may include the framework region H-FR4, wherein the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 28: WGQGTLVTVSS.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application 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 45 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, where 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
  • 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 NO: 1, 9, 12, 14 and 32.
  • Antigen binding protein described herein may comprise a light chain variable region VH, wherein said VH may comprise SEQ ID NO: amino acid sequence of 54: QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX 44 X 45 LEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS, wherein X 44 is K or N, and, X 45 is G or K.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • 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 protein described in the present application may comprise a heavy chain constant region, and the heavy chain constant region may be derived from human IgG, for example, human IgG1.
  • the Fc region of human IgG1 can be modified to achieve the desired properties (for example, ADCC KO).
  • the modification may be an amino acid mutation.
  • the modification of IgG1 can be L234A/L235A, that is, according to EU numbering, the amino acids at positions 234 and 235 are respectively mutated from leucine (L) to alanine (A).
  • the heavy chain constant region of the antigen binding protein described in this application may be derived from wild-type human IgG1.
  • the antigen binding protein described in the present application may include a heavy chain constant region, which may include 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 include at least one CDR in the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 64.
  • the isolated antigen binding protein described in the present application may include at least one CDR in the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 63.
  • the isolated antigen binding protein may comprise at least one CDR in the light chain variable region VL, and the VL may comprise any one of SEQ ID NO: 2, 10, 11, 13, 33 and 34 The amino acid sequence shown.
  • the VL of the antigen binding protein may include LCDR1, LCDR2, and LCDR3.
  • the antigen binding protein described in the present application 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 6 and 52.
  • the antigen binding protein described in the present application may include LCDR1, wherein the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6: QHVSTA.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may include LCDR2, wherein the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7: SAS.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise LCDR3, wherein the LCDR3 may comprise the amino acid sequence shown 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 8 and 53.
  • the antigen binding protein described in the present application may include LCDR3, wherein the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 8: QQHYITPYT.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may also include framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
  • the antigen binding protein described in the present application may comprise the framework region L-FR1, wherein the L-FR1 may comprise the amino acid sequence shown 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; where 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, and 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 15, 16, 44, and 45.
  • the antigen binding protein described in the present application may include the framework region L-FR1, wherein the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 67: DIQMTQSPSSLSASVGDRVTITCX 24 AS; wherein X 24 is K or R.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 15 and 16.
  • the antigen binding protein described in the present application may include the framework region L-FR2, wherein the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 70: X 1 X 2 WYQQKPGX 10 X 11 PKLLIX 17 ; where X 1 Is L or V, X 2 is A or N, X 10 is K or Q, X 11 is A or S, and X 17 is H or Y.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 17, 18, 46, and 47.
  • the antigen binding protein described in the present application may comprise the framework region L-FR2, wherein the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69: X 1 X 2 WYQQKPGKAPKLLIY; wherein X 1 is L or V, And X 2 is A or N.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 17 and 18.
  • the antigen binding protein described in the present application may comprise the framework region L-FR3, wherein the L-FR3 may comprise the amino acid sequence shown in 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; where 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 19-21 and 48-50.
  • the antigen binding protein described in the present application may comprise the framework region L-FR3, wherein the L-FR3 may comprise the amino acid sequence shown 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 19-21.
  • the antigen binding protein described in the present application may comprise the framework region L-FR4, wherein the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 73: FGX 3 GTKLEIK, where X 3 is G or Q.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR4 may include the amino acid sequence shown in any one of SEQ ID NO: 22 and 51.
  • the antigen binding protein described in the present application may include the framework region L-FR4, wherein the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 22: FGQGTKLEIK.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise a light chain variable region VL, wherein the VL may comprise the amino acid sequence shown in SEQ ID NO: 64: DIX 3 MTQSX 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, where X 3 is Q or V, X 8 is H or P, X 9 is K or S, X 10 is F or S, and X 11 is L or M, X 12 is F or S, X 13 is A or T, X 15 is
  • 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 in the present application may include a light chain variable region VL, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 63: DIQMTQSPSSLSASVGDRVTITCX 24 ASQHVSTAX 33 X 34 WYQQKPGKAPKLLIYSASYX 54 X 55 SGVPSRFSGSX 66 SGTDFTLTISSLQPEDFATYYCQQHYKLEITFGGTLE 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.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • 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 in the present application may include a light chain constant region, and the light chain constant region may include a human light chain constant region sequence, for example, may include a human kappa light chain constant region.
  • the light chain constant region of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 31.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 may include The amino acid sequence shown in SEQ ID NO:56, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:57.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 It may include the amino acid sequence shown in any one of SEQ ID NOs: 4 and 42, and the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 5 and 43.
  • the isolated antigen binding protein may comprise the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2 and HCDR3, and the HCDR1, the HCDR2 and the HCDR3 may respectively comprise SEQ ID NO: 3, The amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: 5.
  • the isolated antigen binding protein may comprise the CDRs of the variable region of the antibody heavy chain—HCDR1, HCDR2 and HCDR3, and the HCDR1, the HCDR2 and the HCDR3 may respectively comprise SEQ ID NO: 3, The amino acid sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 65, and the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 66.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2, and LCDR3, and the LCDR1 may include SEQ ID NO: 6 and 52.
  • the amino acid sequence, the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 7
  • the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 8 and 53.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2, and LCDR3, and the LCDR1, the LCDR2, and the LCDR3 may respectively include SEQ ID NO: 6, The amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.
  • the isolated antigen binding protein may include the CDRs of the variable region of the antibody light chain-LCDR1, LCDR2, and LCDR3, and the LCDR1, the LCDR2, and the LCDR3 may respectively include SEQ ID NO: 52, The amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 53.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 may include SEQ ID NO.
  • the amino acid sequence shown in SEQ ID NO: 56, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 57
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 65
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 7, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 66.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 may include SEQ
  • the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 7 and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 8 and 53.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3
  • SEQ ID NO: 3 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 may be included in sequence.
  • the isolated antigen binding protein described in this application may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the The LCDR3 may respectively include 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 sequence.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 55, and the VL may include The amino acid sequence shown in SEQ ID NO: 64.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, and the VH may include SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 12.
  • the amino acid sequence shown in any one of SEQ ID NO: 32 and SEQ ID NO: 14, and the VL may include 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 shown in any one of the amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 54, and the VL may include The amino acid sequence shown in SEQ ID NO: 63.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, and the VH may include SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO:
  • the amino acid sequence shown in any one of 14, and the VL may include the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 13.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO: 9
  • the VL may include SEQ ID NO : The amino acid sequence shown in 10.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO: 9
  • the VL may include SEQ ID NO : The amino acid sequence shown in 11.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO: 12
  • the VL may include SEQ ID NO : The amino acid sequence shown in 13.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO: 14
  • the VL may include SEQ ID NO : The amino acid sequence shown in 11.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO:1
  • the VL may include SEQ ID NO : The amino acid sequence shown in 2.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO: 32
  • the VL may include SEQ ID NO : The amino acid sequence shown in 34.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include the amino acid sequence shown in SEQ ID NO: 32
  • the VL may include SEQ ID NO : The amino acid sequence shown in 33.
  • the isolated antigen binding protein described in this application may also 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 a human kappa light chain constant region .
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 9, and the VL may include SEQ ID NO : 10; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 29, and The light chain constant region may include the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as HB0030.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 9, and the VL may include SEQ ID NO : 11; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 29, and The light chain constant region may include the amino acid sequence shown in SEQ ID NO: 31.
  • the isolated antigen binding protein may be referred to as HB0031.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 12, and the VL may include SEQ ID NO The amino acid sequence shown in: 13; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 29, and The light chain constant region may include the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as HB0032.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 14, and the VL may include SEQ ID NO The amino acid sequence shown in: 11; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, and the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 29, and The light chain constant region may include the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as HB0033.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO:1, and the VL may include SEQ ID NO : 2; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, and the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 30, and The light chain constant region may include the amino acid sequence shown in SEQ ID NO: 31.
  • the isolated antigen binding protein may be referred to as 900424.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 32, and the VL may include SEQ ID NO The amino acid sequence shown in: 34; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, and the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 30, and The light chain constant region may include the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as 900423.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 32, and the VL may include SEQ ID NO : 33; and, the isolated antigen binding protein may include a heavy chain constant region and a light chain constant region, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 30, and The light chain constant region may include 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 this 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 chain variable region may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 56; the HCDR3 may include SEQ The amino acid sequence shown in ID NO: 57, the light chain variable region of the reference antibody may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 65; the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 7; the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 66.
  • the isolated antigen-binding protein described in this 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 chain variable region may include HCDR1, HCDR2, and HCDR3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 4; and the HCDR3 may include SEQ ID NO: 5, the light chain variable region of the reference antibody may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 6; the LCDR2 may include The amino acid sequence shown in SEQ ID NO: 7; the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 8.
  • the protein, polypeptide, and/or amino acid sequence involved in this application should also be understood to include at least the following range: a variant or homologue that has the same or similar functions as the protein or polypeptide.
  • the variant may be one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (for example, the antigen binding protein described in the present application).
  • Protein or peptide may include at least one, such as 1-30, 1-20, or 1-10, and another example, 1, 2, 3, 4, or 5 amino acid substitutions.
  • the functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion, or addition).
  • the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide before the change.
  • the substitution may be a conservative substitution.
  • a part of the amino acid sequence of the antigen binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific category.
  • both the variable region and constant part of an antibody can be derived from the variable region and constant region of an antibody of an animal species (such as human).
  • the homologue may be at least about 85% (for example, having at least about 85% of the amino acid sequence of the protein and/or the polypeptide (for example, the antigen binding protein described in this application) %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology Protein or peptide.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology" can be calculated in the following way: the two sequences to be aligned are compared in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to generate the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu,
  • the alignment to determine the percent sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA for a description of the FASTA algorithm, please refer to WRPearson and DJ Lipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • the antigen binding protein (eg, TIGIT antibody) described in this application can specifically bind to the TIGIT antigen.
  • Antigen binding proteins eg, antibodies
  • TIGIT antigens can generally bind to TIGIT with a KD value of about 1 nM or higher (eg, 1 nM, 100 pM, 10 pM, 2 pM, or 1 pM), but not to the lack of TIGIT sequences Of other proteins.
  • an antibody that "specifically binds" TIGIT does not bind human CD226, human CD155, and human CD112.
  • the antigen binding protein (for example, antibody) described in this application can specifically bind to TIGIT antigen or its labeled form (for example, fluorescently labeled TIGIT antigen), but will not bind to other proteins lacking TIGIT epitopes. Whether an antigen binding protein (e.g., antibody) binds to the TIGIT antigen can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET). In some cases, the TIGIT antibodies described in this application can also cross-react with monkey and/or mouse TIGIT. For example, it can be detected by flow analysis technology and enzyme-linked immunoassay. As used herein, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.
  • the antigen binding protein (eg, TIGIT antibody) described in this application can inhibit the binding of TIGIT to CD155 ligand.
  • the blocking experiment can be detected by a competition method, for example, the antigen binding protein (for example, TIGIT antibody) and the antigen (or cells that can express the antigen) and the ligand of the antigen (or, the cell that expresses the ligand)
  • the intensity for example, fluorescence intensity or concentration
  • the IC50 of the antigen binding protein (eg, TIGIT antibody) described in the present application to block the binding of TIGIT antigen and CD155 ligand is about 0.1 ⁇ g/ml-0.05 ⁇ g/ml.
  • the application also provides one or more isolated nucleic acid molecules.
  • the one or more nucleic acid molecules may encode the antigen binding protein described in this application.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode part of it (for example, HCDR1-3, LCDR1-3, VL, VH, light chain Or one or more of the heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody and its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides. Overlapping extension PCR. For specific operations, please refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
  • this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters can include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part can be fused with a certain part of a gene promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked 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.
  • the vector is an expression vector.
  • the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application.
  • each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in this application can be introduced into the host cell, such as a eukaryotic cell, such as a plant-derived cell, fungus, or yeast cell.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application provides a method for preparing the antigen binding protein.
  • the method may include culturing the host cell described in the present application under conditions that allow the expression of the antigen binding protein. For example, it is possible to use an appropriate medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.
  • any method suitable for the production of monoclonal antibodies can be used to produce the antigen binding proteins of the present application (for example, anti-TIGIT antibodies).
  • animals can be immunized with linked or naturally occurring TIGIT homodimers or fragments thereof.
  • Appropriate immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more approaches can be used.
  • TIGIT can be used as an immunogen (antigen) to produce non-human antibodies specific to TIGIT and to screen the biological activity of the antibodies.
  • the eliciting 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 natural sources or produced in genetically modified cells.
  • the DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in source.
  • Suitable genetic vectors can be used to express DNA encoding immunogens, including but not limited to adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, materials, and non-viral vectors.
  • Example 1 An exemplary method of producing the anti-human TIGIT antibody of the present application is described in Example 1.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody, and the IgG1 subtype is used.
  • the optimization of the necessary constant domain sequence can be achieved by screening antibodies with the biological assays described in the Examples below to produce the desired biological activity.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof can be used in the compounds and methods of this application.
  • Example 2 An exemplary method of humanizing the anti-human TIGIT antibody of the present application is described in Example 2.
  • sequence of the DNA molecule of the antigen binding protein or its fragments of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can also be fused together to form a single chain antibody.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences. The nucleic acid molecule can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • This application also relates to vectors containing the above-mentioned appropriate nucleic acid molecules and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • animal cells may include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
  • the steps of transforming host cells with recombinant DNA described in this application can be performed by techniques well known in the art.
  • the obtained transformant can be cultured by conventional methods, and the transformant expresses the polypeptide encoded by the nucleic acid molecule of the present application.
  • the host cell used it is cultured in a conventional medium under suitable conditions.
  • the transformed host cells are cultured under conditions suitable for the expression of the antigen binding protein of the present application.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antigen-binding protein of the present application is purified by conventional separation and purification methods well-known to the person.
  • the obtained monoclonal antibody can be identified by conventional means.
  • the binding specificity of 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)).
  • FACS flow cytometry
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the composition may be a pharmaceutical composition, which contains the antigen binding protein or fusion protein or ADC or corresponding CAR-T cell of the present application, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH can usually be about 5-8, for example, the pH can be about 6-8, and the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated 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 a cell by a nucleotide sequence for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition described in the present application can be directly used to bind TIGIT protein molecules, and thus can be used to prevent and treat TIGIT-related diseases.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present application may contain a safe and effective amount (such as 0.001-99 wt%, 0.01-90 wt%, or 0.1-80 wt%) of the antigen binding protein described in the present application and a pharmaceutically acceptable adjuvant (which may include a carrier Or excipients).
  • a pharmaceutically acceptable adjuvant which may include a carrier Or excipients.
  • Such carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition described in the present application can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic 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.
  • the antigen binding protein or pharmaceutical composition described herein can be formulated, administered, and administered in a manner consistent with good medical practice.
  • the considerations in this situation 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 the medical practitioner .
  • Therapeutic agents e.g., anti-TIGIT antibodies
  • the effective amount of such other agents depends on the amount of therapeutic agent (e.g., anti-TIGIT antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above.
  • These agents can generally be used in any dosage that is empirically/clinically determined to be appropriate and through any route that is empirically/clinically determined to be appropriate. Compared with a single treatment, the dose of the antibody administered in the combination treatment can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.
  • the application provides the use of the antigen binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of medicines.
  • the medicine is used to prevent, alleviate and/or treat TIGIT-related diseases.
  • the TIGIT-related disease may be a T cell dysfunction disorder. T cell dysfunction is manifested in the exhaustion of T cells, which is achieved by strengthening NK cells and activating T cells, and enhancing the immune activity of the body to achieve the treatment or delay or alleviation of diseases.
  • the TIGIT-related disease may be tumor, cancer or infectious disease.
  • the TIGIT-related disease may be a CD155-positive or PVR-positive tumor, cancer, immune disease, or infectious disease, including tumor, cancer, immune disease, or infectious disease.
  • the tumor may be selected from the group consisting of breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, kidney cancer, melanoma, T lymphoma, pancreatic cancer.
  • the tumor is colon cancer.
  • the antigen binding protein described in the present application can inhibit tumor growth in animal models of tumors selected from the group consisting of breast cancer, colon cancer, liver cancer, lymphoma, chondrosarcoma, multiple myeloma, lung cancer, kidney cancer, and melanoma , T lymphoma and pancreatic cancer.
  • the antigen binding protein of the present application can inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor comprises colon cancer.
  • the tumor or cancer is a tumor or cancer with abnormal TIGIT expression.
  • This application also provides the method for detecting the expression of TIGIT in a biological sample as described below.
  • the method includes contacting a biological sample with the antigen-binding protein described in this application under conditions that allow the antigen-binding protein to bind to TIGIT, and detecting whether there is formation between the antigen-binding protein and TIGIT. Complex.
  • the tumor or cancer is a tumor or cancer in which the expression of TIGIT is increased compared to a non-tumor or cancer sample.
  • the antigen-binding protein described in this application can be used in, for example, immunoassays, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blotting), flow cytometry (for example, FAGS ) And enzyme-linked immunosorbent assay (ELISA).
  • immunoassays including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blotting), flow cytometry (for example, FAGS ) And enzyme-linked immunosorbent assay (ELISA).
  • IHC immunohistochemistry
  • IF immunofluorescence
  • IF immunoblotting
  • flow cytometry for example, FAGS
  • ELISA enzyme-linked immunosorbent assay
  • the application also provides the use of the antigen binding protein in a method for diagnosing a subject suffering from a disorder (for example, cancer or immune dysfunction), the method comprising: binding a sample to the antigen of the application The protein contacts and detects the presence of the bound antigen binding protein and/or fusion protein to determine the presence or expression level of TIGIT in the sample obtained from the subject.
  • a disorder for example, cancer or immune dysfunction
  • the antigen binding protein eg, TIGIT antibody
  • TIGIT antibody can slow tumor growth.
  • the antigen binding protein of the present application can be used for detection applications, for example, for testing samples, thereby providing diagnostic information.
  • samples include cells, tissue samples and biopsy specimens.
  • biopsy used in this application shall include all types of biopsy known to those skilled in the art. Therefore, the biopsy used in this application may include, for example, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
  • the samples used in this application include fixed or preserved cell or tissue samples.
  • the application also provides a kit containing the antigen binding protein of the application.
  • the kit may also include a container, instructions for use, buffers, and the like.
  • the original binding protein of the present application can be immobilized on a detection plate.
  • the final monoclonal hybridoma cell line obtained has high affinity, can have binding activity with both humans and monkeys, has blocking function for the two ligands of TIGIT, CD155 and CD122, and has biological activity.
  • the hybridoma cell lines in Table 1 above were humanized.
  • the cDNA sequence of the variable region of the mouse antibody expressed by the hybridoma cell line was cloned.
  • the SMARTer 5'RACE synthesis kit (TAKARA, #634859) was used to synthesize the variable region gene-specific cDNA of the heavy and light chains according to the instructions.
  • the primers are designed to add appropriate leader sequences to the variable region cDNA of the heavy chain and light chain, respectively, so that the resulting PCR product can be cloned by a seamless cloning method.
  • the existing recombinant antibody expression heavy chain vector pHB-Fc and light chain vector pHB-C ⁇ contains the human IgG1 heavy chain constant region gene sequence, in which CH 2 carries the L234A and L235A (Eu code) mutations with the antibody ADCC knock out (KO) effect; the pHB-C ⁇ vector contains the human kappa light chain Constant region gene sequence.
  • the PCR amplification products of the heavy and light chain variable regions were cloned into an expression vector using In-fusion cloning reagent (TAKARA, #639650), and transformed into E. coli DH5 ⁇ competent cells of E. coli (Ebiotics Technology, #FYE607) -80VL). By selecting monoclonal colonies for Sanger sequencing, the antibody variable region sequence was obtained by analysis.
  • the sequence of the variable region of the anti-TIGIT antibody expressed by B3/29F6 is as follows:
  • the underlined regions are CDRs (as defined by IMGT, the sequences are as follows):
  • IgG1 wild type is chimeric antibody and ADCC KO type chimeric antibody share the same light chain carrier, the difference is that the 234 and 235 amino acids of heavy chain CH 2 on the carrier containing the human heavy chain constant region sequence are used, ADCC KO type It is A234/A235, and the wild type is L234/L235.
  • the method of IgG1 wild-type chimeric heavy chain expression vector is the same as that of the expression vector construction method described in 2.1, that is, the heavy chain variable region gene is amplified by PCR with primers containing the leader sequence, and then cloned into humans by seamless ligation. On the vector of the heavy chain constant region sequence (CH2 is L234/L235), the construction of the IgG1 wild-type chimeric expression vector is completed.
  • the expression vector obtained in 2.1 and 2.2 was amplified by Escherichia coli, and a sufficient amount of plasmid was prepared with endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) for transient transfection and expression of chimeric antibody .
  • the host cell used for expression is CHO-S cell (Invitrogen, #R80007).
  • the two prepared heavy chain carriers were mixed with the light chain carrier to form a liposome complex with polyetherimide (PEI, Polysciences, #24765-1), and then transfected into CHO-S cells. Incubate in an incubator for 5-7 days.
  • the cell culture supernatant was collected by centrifugation, and purified by a Protein A affinity chromatography column to obtain ADCC KO type human-mouse chimeric antibody (number 900424) and IgG1 wild-type human-mouse chimeric antibody (protein number 900445).
  • VH and VH sequences of the chimeric antibody obtained by the above method are as follows:
  • the amino acid sequence of the heavy chain constant region of 900424 is shown in SEQ ID NO: 30, the amino acid sequence of the heavy chain constant region of 900423 is shown in SEQ ID NO: 30, and the amino acid sequence of the heavy chain constant region of 900428 is shown in SEQ ID NO: 30.
  • the light chain constant regions are all human kappa light chain constant regions (SEQ ID NO: 31).
  • variable region sequence of the antibody was compared with the available sequences in the NCBI IgBlast database.
  • FR regions human-derived framework regions
  • the modification sites are designed, and the variable regions of the heavy and light chains of the chimeric antibody are respectively designed for humanized mutations.
  • PCR technology amplifies and constructs humanized point mutation antibody expression plasmid.
  • the humanized point mutation antibody expression plasmids were expressed in CHO-S cells respectively, and the humanized antibody protein was obtained after purification.
  • ELISA, Biacore and flow cytometry and other detection methods to screen the humanized antibody's receptor binding ability, functional inhibitory activity and ADCC effect and other indicators, four humanized anti-TIGIT antibodies with excellent performance were obtained.
  • the VH and VL sequences of the obtained humanized anti-TIGIT antibody are shown below:
  • the underlined area is CDRs (defined according to IMGT), 900461, 900464, 900466 and 900476 are the four humanized antibody protein numbers respectively, and the corresponding item numbers are HB0030, HB0031, HB0032, HB0033.
  • the heavy chain constant region amino acid sequence of HB0030 is shown in SEQ ID NO: 29
  • the heavy chain constant region amino acid sequence of HB0031 is shown in SEQ ID NO: 29
  • the heavy chain constant region amino acid sequence of HB0032 is shown in SEQ ID NO: 29
  • the amino acid sequence of the heavy chain constant region of HB0033 is shown in SEQ ID NO: 29.
  • the light chain constant regions are all human kappa light chain constant regions (SEQ ID NO: 31).
  • All chimeric antibodies and humanized antibodies are diluted to 20 ⁇ g/ml with 1% BSA-containing PBS solution (1%BSA/PBS), 20 ⁇ L per well is added to the 96-well U-shaped plate, and the negative control is set simultaneously (only add 1%BSA/PBS).
  • the 96-well U-shaped plate after the reaction was resuspended in 1% BSA/PBS, centrifuged (300g ⁇ 3min) to discard the supernatant, washed once, and added 1:200 diluted PE-goat anti-human-Fc (Jackson ImmunoResearch, #109-115-098), react at room temperature and avoid light for 15 minutes; resuspend the 96-well U-shaped plate after the reaction in 1% BSA/PBS, centrifuge (300g ⁇ 3min) to discard the supernatant, and wash 3 times in this way.
  • the well was resuspended in 100 ⁇ L of 1% BSA/PBS, and the fluorescence intensity of the PE channel was detected with a flow cytometer (BD, #CantoII).
  • the results of the binding activity of the chimeric antibody are shown in Figure 1 and Table 3, and the results of the binding activity of the humanized antibody are shown in Figure 2 and Table 4.
  • 900324 is the anti-human TIGIT antibody Tiragolumab from Genentech that is transiently expressed in CHO cells. Its light and heavy chains can be The sequence of the variable region is the same as that of the antibody 10A7 in the patent application WO2015009856A2.
  • the results show that both the chimeric antibody and the humanized antibody have good affinity with the cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Huabo Biological), and the affinity is similar.
  • Antibody 900324 HB0030 HB0031 HB0032 HB0033 EC 50( ⁇ g/mL) 0.08994 0.04661 0.05806 0.07847 0.0514
  • the antigen huCD155-moFc (ACROBiosystems, #CD5-H5254) was diluted to 40 ⁇ g/ml with 1% BSA in PBS solution (1%BSA/PBS), and 10 ⁇ L per well was added to a 96-well U-shaped plate. TIGIT antibody is mixed evenly at a volume ratio of 1:1, and the positive control is set simultaneously (only CD155-moFc is added).
  • the 96-well U-shaped plate after the reaction was resuspended in 1% BSA/PBS, centrifuged (300g ⁇ 3min) to discard the supernatant, washed once, and added 1:300 diluted Alexa488-goat anti-mouse-Fc (Jackson ImmunoResearch, #115-545-071), react at room temperature and avoid light for 15 minutes; resuspend the 96-well U-shaped plate after the reaction in 1% BSA/PBS, centrifuge (300g ⁇ 3min) to discard the supernatant, and wash 3 times in this way.
  • the well was resuspended in 100 ⁇ L of 1% BSA/PBS, and the fluorescence intensity of the FITC channel was detected with a flow cytometer (BD, #CantoII).
  • Antibody 900324 HB0030 HB0031 HB0032 HB0033 IC 50 ( ⁇ g/mL) 0.1349 0.05733 0.06653 0.0919 0.05598
  • This test uses the SPR method to determine the antigen-antibody binding kinetics and affinity.
  • Use BIOCORE GE, #Biacore8K.
  • Sereis Sensor Chip Protein A chip GE, #29-1275-56) equilibrate at room temperature for 20-30 minutes, and load the chip into the instrument. Dilute the antibody sample to the experimental working concentration with equilibration buffer, and seal it at 2-8°C for later use.
  • HBS-EP 10x
  • His-Tag human TIGIT protein
  • the sample was analyzed by the capture method multi-cycle kinetics program, the corresponding analysis program was selected to analyze the data, and it was confirmed that there was no obvious reference binding, and the Kinetics, 1:1 binding modle was selected, and the kinetic parameters of the sample were obtained by fitting analysis.
  • mice were used, CT26.WT cells in logarithmic growth phase were collected, culture medium was removed and washed twice with PBS, and then inoculated (the survival rate of CT26.WT cells before and after tumor bearing respectively) They are: 98.57% and 98.28%), inoculation amount: 5 ⁇ 10 5 /100 ⁇ L/head.
  • the mice were randomly grouped according to the tumor volume, with 8 mice in each group. The grouping day is defined as D0 day, and the drug is administered on D0, D3, D7, D10, D14, and D17. D23 observes the tumor size and weighs the mice.
  • the anti-TIGIT antibody HB0030 of the present application has a better tumor suppressing effect compared with the control antibody Tiragolumab.
  • mice in all groups were treated 24 days after inoculation, and there was no significant change in the body weight of the mice.
  • 900541 is HB0030 with no ADCC effect with L234A/L235A mutation in the Fc region, and the other two are samples of different batches of HB0030.
  • physiological saline was used as a negative control to evaluate the anti-tumor effects of the above three drugs in mouse colon cancer cell CT26.WT transplanted humanized mouse BALB/c-hPD1/hTIGIT.

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Abstract

提供了一种分离的抗原结合蛋白，该抗原结合蛋白包含重链可变区VH中的至少一个CDR，该VH包含SEQ ID NO:55所示的氨基酸序列。还提供了抗TIGIT的抗体、其制备方法和应用。该抗原结合蛋白能够特异性结合TIGIT抗原，阻断TIGIT与其配体的结合，并且能够用于制备预防或治疗TIGIT相关的疾病的药物。

Description

抗TIGIT的抗体、其制备方法和应用 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向于TIGIT的抗原结合蛋白、其制备方法和应用。
背景技术
正常情况下，T细胞依赖抗原提呈细胞(Antigen-presenting cells，APCs)提呈的外源或内源抗原-MHC-I/II复合物与T细胞(CD4 +、CD8 +)表面受体(T Cell Receptor，TCR)结合以及额外的共刺激(Costimulatory)信号来充分激活，然后通过其他效应细胞对表达相关抗原的细胞进行杀伤。相反的是，T细胞还受到共抑制(Coinhibitory)信号调节，后者对T细胞的活性、增殖、存活等具有负面调节作用，也可维持自身耐受，防止过度的自身免疫反应，减小免疫应答所致的组织损伤。在肿瘤细胞微环境中，T细胞的激活与抑制处于异常模式，目前，其正常化被广泛视为肿瘤治疗的重要手段。
TIGIT(T cell Ig and ITIM domain，也称为WUCAM、Vstm3或VSIG9)是脊髓灰质炎病毒受体(PVR)/Nectin家族的成员。它由细胞外免疫球蛋白可变区(IgV)结构域，1型跨膜结构域和具有经典免疫受体酪氨酸抑制基序(ITIM)和免疫球蛋白酪氨酸尾(ITT)基序的细胞内结构域组成。TIGIT在淋巴细胞中表达，特别是在效应和调节性CD4 +T细胞，滤泡辅助CD4 +T细胞，效应CD8 +T细胞和自然杀伤(NK)细胞中高表达。TIGIT基因位于人类第16号染色体，编码由244个氨基酸组成的I型跨膜蛋白。人TIGIT分子胞膜外区长141个氨基酸，有1个免疫球蛋白V样结构域；跨膜区23个氨基酸；胞质区较短，有80个氨基酸，具有1个PDZ结合结构域和1个ITIM模体。
CD155(也叫PVR、Necl5或Tage4)是TIGIT的高亲和力配体。肿瘤表面高表达的CD155一旦与NK和T细胞表面的TIGIT结合，它们对肿瘤细胞的杀伤作用就会被抑制。TIGIT可以通过阻止肿瘤细胞的初始死亡和释放肿瘤抗原来抑制NK细胞效应；抑制树突细胞协同刺激能力，导致癌抗原呈递减少和抗炎细胞因子如IL-10增加，TIGIT还可以诱导其他细胞如肿瘤细胞的PVR信号传导；TIGIT可以直接抑制CD8 +T细胞效应，或者TIGIT +Treg可以抑制CD8 +T细胞，防止癌细胞的清除(Manieri et al.,Inhibition of the Cancer Immunity Cycle by TIGIT.2016)。
大量的临床前研究结果表明，TIGIT将成为癌症乃至其他疾病患者的合适靶标，因此，鉴于TIGIT在各类相关疾病中作用和功能，本领域仍然需要开发适于治疗患者的改善的抗TIGIT特异性抗体。
发明内容
本申请提供了一种分离的抗原结合蛋白可以包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:55所示的氨基酸序列。所述分离的抗原结合蛋白也可包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:64所示的氨基酸序列。所述分离的抗原结合蛋白具有下述性质中的一种或多种：1)能够以1×10 -10M或更低(例如，8×10 -11M、5×10 -11M、2×10 -11M、1×10 -11M或更低)的KD值结合TIGIT蛋白，其中所述KD值通过表面等离子体共振法测定；2)在FACS测定中，能够阻断CD155与TIGIT的结合；和3)能够抑制肿瘤生长和/或肿瘤细胞增殖。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:55所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白具有下述性质中的一种或多种：1)能够以1×10 -10M或更低的KD值结合TIGIT蛋白，其中所述KD值通过表面等离子体共振法测定；2)在FACS测定中，能够阻断CD155与TIGIT的结合；和3)能够抑制肿瘤生长和/或肿瘤细胞增殖。
在某些实施方式中，所述分离的抗原结合蛋白与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体包含重链可变区和轻链可变区，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列；所述HCDR2包含SEQ ID NO:56所示的氨基酸序列；所述HCDR3包含SEQ ID NO:57所示的氨基酸序列，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:65所示的氨基酸序列；所述LCDR2包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3包含SEQ ID NO:66所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源 抗体。
在某些实施方式中，所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:56所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:4和42中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:57所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:5和43中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，且所述分离的抗原结合蛋白包含选自下述的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:3，HCDR2：SEQ ID NO:4，和HCDR3：SEQ ID NO:5；以及
(2)HCDR1：SEQ ID NO:3，HCDR2：SEQ ID NO:42，和HCDR3：SEQ ID NO:43。
在某些实施方式中，所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:65所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NO:6和52中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO:66所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1、LCDR2和LCDR3，且所述分离的抗原结合蛋白包含选自下述的任意一组氨基酸序列：
(1)LCDR1：SEQ ID NO:6，LCDR2：SEQ ID NO:7，和，LCDR3：SEQ ID NO:8；以及
(2)LCDR1：SEQ ID NO:52，LCDR2：SEQ ID NO:7，和，LCDR3：SEQ ID NO:53。在某些实施方式中，所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
在某些实施方式中，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:68所示的氨基酸序列。
在某些实施方式中，所述L-FR1包含SEQ ID NO:15、16、44和45中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述L-FR2包含SEQ ID NO:17、18、46和47中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述L-FR3包含SEQ ID NO:19、20、21、48、49和50中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中，所述L-FR4包含SEQ ID NO:22和51中任一项所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:64所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:2、10、11、13、33和34中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体轻链恒定区，且所述抗体轻链恒定区来自人κ轻链恒定区，且所述人κ轻链恒定区包含SEQ ID NO:31所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在某些实施方式中，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:23和35中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:24、25、26、36和37中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:61所示的氨基酸序列。
在某些实施方式中，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:27、38和39中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:62所示的氨基酸序列。
在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4 包含SEQ ID NO:28、40和41中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:55所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:1、9、12、14和32中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
在某些实施方式中，所述抗体重链恒定区包括人IgG1恒定区，且人IgG1恒定区包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含重链可变区VH和轻链可变区VL，且所述分离的抗原结合蛋白包含选自下述的任意一组氨基酸序列：(1)VH：SEQ ID NO:9，VL：SEQ ID NO:10，(2)VH：SEQ ID NO:9，VL：SEQ ID NO:11，(3)VH：SEQ ID NO:12，VL：SEQ ID NO:13，(4)VH：SEQ ID NO:14，VL：SEQ ID NO:11，(5)VH：SEQ ID NO:1，VL：SEQ ID NO:2，(6)VH：SEQ ID NO:32，VL：SEQ ID NO:34，以及(7)
VH：SEQ ID NO:32，VL：SEQ ID NO:33。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码所述的分离的抗原结合蛋白。
另一方面，本申请提供了一种载体，所述载体包含所述的核酸分子。
另一方面，本申请提供了一种细胞，所述细胞包含所述的核酸分子或所述的载体。
另一方面，本申请提供了多肽，所述多肽包含所述的分离的抗原结合蛋白。
另一方面，本申请提供了免疫缀合物，所述免疫缀合物包含所述的分离的抗原结合蛋白或所述的多肽。
另一方面，本申请提供了一种制备所述的分离的抗原结合蛋白的方法，所述方法包括在使得所述的分离的抗原结合蛋白表达的条件下，培养所述的细胞。
另一方面，本申请提供了一种药物组合物，其包含所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的多肽、所述的免疫缀合物和/或所述的细胞，以及任选地药学上可接受的佐剂。
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的多肽、所述的免疫缀合物和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗TIGIT相关疾病。
在某些实施方式中，所述TIGIT相关疾病为T细胞功能障碍性病症。
在某些实施方式中，所述TIGIT相关疾病为肿瘤。在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤选自下组：乳腺癌、结肠癌、肝癌、淋巴瘤、软骨肉瘤、多发性骨髓瘤、肺癌、肾癌、黑色素瘤、T淋巴瘤和胰腺癌。
另一方面，本申请提供了一种抑制CD155与TIGIT结合的方法，所述方法包括施用所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的多肽、所述的免疫缀合物和/或所述的药物组合物。
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的多肽、所述的免疫缀合物和/或所述的药物组合物，其用于预防、缓解或治疗TIGIT相关疾病。
另一方面，本申请提供了一种预防、缓解或治疗TIGIT相关疾病的方法，所述方法包括向有需要的受试者施用所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的多肽、所述的免疫缀合物和/或所述的药物组合物。
在某些实施方式中，所述TIGIT相关疾病为T细胞功能障碍性病症。
在某些实施方式中，所述TIGIT相关疾病为肿瘤。在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤选自下组：乳腺癌、结肠癌、肝癌、淋巴瘤、软骨肉瘤、多发性骨髓瘤、肺癌、肾癌、黑色素瘤、T淋巴瘤和胰腺癌。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是TIGIT嵌合抗体对表达人TIGIT的细胞结合活性。
图2显示的是TIGIT人源化抗体对表达人TIGIT的细胞结合活性。
图3显示的是TIGIT嵌合抗体对人TIGIT与其配体CD155结合的阻断活性。
图4显示的是TIGIT人源化抗体对人TIGIT与其配体CD155结合的阻断活性。
图5显示的是TIGIT抗体在动物体内药效检测。
图6显示的是TIGIT抗体在动物体内与对照抗体Tiragolumab的药效检测比较。
图7显示的是TIGIT抗体在动物体内的药效实验结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请的其他优点及效果。
术语定义
在本申请中，术语“TIGIT”称为“含Ig和ITIM结构域的T细胞免疫受体”，通常为免疫球蛋白的PVR(脊髓灰质炎病毒受体)家族的成员，其可结合PVR/CD155和Nectin-2/CD112。本申请的TIGIT可以指来自任何脊椎动物来源，包括哺乳动物，诸如灵长类(例如人、恒河猴和食蟹猴)和啮齿类(例如小鼠和大鼠)的TIGIT蛋白。该术语涵盖全长的TIGIT或其片段(诸如其缺乏信号肽的成熟片段)，未加工的TIGIT，因细胞中的加工所致的任何形式的TIGIT，以及人工合成的TIGIT。该术语还涵盖TIGIT的变体，例如剪接变体或等位变体。在某些情形中，TIGIT为人TIGIT，人TIGIT的氨基酸序列包含UniProt登录号Q495A1所示的氨基酸序列。在某些情形中，TIGIT为食蟹猴TIGIT，食蟹猴TIGIT的氨基酸序列包含UniProt登录号G7NXM4所示的氨基酸序列。在某些情形中，TIGIT为小鼠TIGIT，小鼠TIGIT的氨基酸序列包含UniProt登录号P86176所示的氨基酸序列。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某 些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“抗体”其以最广泛意义使用，且具体涵盖，但不限于，单克隆抗体(包括包含两条轻链和两条重链的全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人类抗体、嵌合抗体和骆驼化单结构域抗体。“抗体”通常可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)结合。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))、基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)和基于IMGT本体论(IMGT-ONTOLOGY)的概念和IMGT Scientific图表规则的IMGT定义规则。
IMGT指国际ImMunoGeneTics信息系统，一种免疫遗传学和免疫信息学的全球参考数据库(http://www.imgt.org)。IMGT专门研究来自人类和其他脊椎动物的免疫球蛋白(IG)或抗体、T细胞受体(TR)、主要组织相容性(MH)，以及来自脊椎动物和非脊椎动物的免疫球蛋白超家族(IgSF)、MH超家族(MhSF)和免疫系统相关蛋白(RPI)。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“KD”、“K D”可互换地使用，通常是指平衡解离常数，“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。可使用结合速率常数(kon)、解离速率常数(kdis)和平衡解离常数(KD)表示抗原结合蛋白(例如抗体)对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH)下测量，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
在本申请中，术语“参比抗体”通常是指本申请所述抗原结合蛋白与之竞争结合抗原TIGIT的抗体。
在本申请中，术语“ADCC”或“抗体依赖性细胞介导的细胞毒性”通常是指这样一种细胞毒性形式，一些分泌型免疫球蛋白结合到某些细胞毒性效应细胞(例如，NK细胞、嗜中性粒细胞和巨噬细胞)上的Fc受体(FcR)上，使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞。介导ADCC的主要细胞(例如NK细胞)只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FeγRIII(可参见Ravetch and Kinet,Annu.Rev.Immunol.9:457-92(1991)第464页表3)。可以进行体外和/或体内细胞毒性测定法以评估目的分子的ADCC活性，例如，可进行体外ADCC测定法，可参见美国专利号No.5,500,362 或No.5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。或者/另外，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes et al.，PNAS(USA)95:652-656(1998)中所披露的。例如，可以进行Fc受体(FcR)结合测定法以确保抗体缺乏FcγR结合(因此有可能缺乏ADCC活性)，但是保留FcRn结合能力。
ADCC活性可通过修饰Fc区而减少。在某些情形中，影响与Fc受体结合的位点可被除去，例如，除去并非补救受体结合位点的位点。在某些情形中，Fc区可经修饰以除去ADCC位点。ADCC位点是本领域已知的，关于IgG1的ADCC位点，参见例如，Sarmay et al.(1992)Molec.Immunol.29(5):633-9。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。本申请所述的分离的抗原结合蛋白通常不结合非TIGIT抗原的抗原。
在本申请中，术语“分离的核酸分子”或“分离的多核苷酸”通产是指基因组、mRNA、cDNA或合成来源的DNA或RNA或其一定组合，其不与在自然界中发现的多核苷酸的全部或一部分缔合，或连接至其在自然界中不连接的多核苷酸。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA 的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“药学上可接受的佐剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是PH缓冲水溶液。生理学可接受载体的例子可包括缓冲剂，诸如磷酸盐、柠檬酸盐和其它有机酸；抗氧化剂，包括抗坏血酸；低分子量(少于约10个残基)多肽，蛋白质，诸如血清清蛋白，明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，诸如甘氨酸，谷氨酰胺，天冬酰胺，精氨酸或赖氨酸；单糖，二糖和其它碳水化合物，包括葡萄糖，甘露糖或糊精；螯合剂，诸如EDTA；糖醇，诸如甘露醇或山梨醇；成盐反荷离子，诸如钠；和/或非离子表面活性剂，诸如TWEEN TM，聚乙二醇(PEG)和PLURONICS TM。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括TIGIT结合物与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本申请的任何一种TIGIT抗原结合蛋白及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症 状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。治疗的期望效果包括降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。例如，若一种或多种与癌症有关的症状是减轻或消除的，包括但不限于，降低(或破坏)癌细胞增殖，减少源自疾病的症状，提高那些患有疾病的个体的生命质量，降低治疗疾病需要的其它药物的剂量，延迟疾病的进展，和/或延长个体存活，则个体得到成功“治疗”。
在本申请中，术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，以及所有癌前(pre-cancerous)和癌性细胞和组织。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供了一种分离的抗原结合蛋白，其可以包含重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:55所示的氨基酸序列。
一方面，本申请提供了一种分离的抗原结合蛋白，其可以包含重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:1、9、12、14和32中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的VH可包含HCDR1、HCDR2和HCDR3。
本申请所述的抗原结合蛋白可以包含HCDR1，其中所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列：GYSITSDYA。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含HCDR2，其中所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列：IX 2X 3SGX 6X 7，其中，X 2为S或T，X 3为S或Y，X 6为A或S，且X 7为P或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述HCDR2可以包含SEQ ID NO:4和42中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含HCDR2，其中所述HCDR2可包含SEQ ID NO:4所示的氨基酸序列：ITSSGST。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含HCDR3，其中所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列：AX 2LX 4X 5X 6X 7YX 9X 10AMDY，其中，X 2为R或S，X 4为D或G，X 5为F或T，X 6为D或G，X 7为N或Y，X 9为G或不存在，X 10为G或不存在。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述HCDR3可以包含SEQ ID NO:5和43中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含HCDR3，其中所述HCDR3可包含SEQ ID NO:5所示的氨基酸序列：ARLDFGNYGGAMDY。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白还可以包含框架区H-FR1、H-FR2、H-FR3和H-FR4。
本申请所述的抗原结合蛋白可以包含框架区H-FR1，其中所述H-FR1可包含SEQ ID NO:58所示的氨基酸序列：X 1VQLQESGPGLVKPSX 16X 17LSLTCTVX 25，其中，X 1为D或Q，X 16为E或Q，X 17为S或T，且X 25为S或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR1可以包含SEQ ID NO:23和35中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR1，其中所述H-FR1可包含SEQ ID NO:23所示的氨基酸序列：QVQLQESGPGLVKPSETLSLTCTVS。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR2，其中所述H-FR2可包含SEQ ID NO:60所示的氨基酸序列：WX 2WIRQX 7PGX 10X 11X 12EWX 15GY；其中，X 2为I或N，X 7为F或P，X 10为K或N，X 11为G、R或K，X 12为L或V，且，X 15为I或M。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR2可包含SEQ ID NO:24-26和36-37中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR2，其中所述H-FR2可包含SEQ ID NO:59所示的氨基酸序列：WIWIRQPPGX 10X 11LEWIGY；其中，X 10为K或N，X 11为G或K。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR2可包含SEQ ID NO:24-26中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR3，其中所述H-FR3可包含SEQ ID NO:61所示的氨基酸序列： X 1YNPSLKSRX 10X 11X 12X 13X 14DTSKNQFX 22LX 24LX 26X 27VTX 30X 31DTATYYC，其中，X 1为R、S或Y，X 10为I或V，X 11为S或T，X 12为F或I，X 13为S或T，X 14为R或V，X 22为F或S，X 24为K或Q，X 26为S或T，X 27为F或S，X 30为A或T，且，X 31为A或E。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR3可包含SEQ ID NO:27、38和39中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR3，其中所述H-FR3可包含SEQ ID NO:27所示的氨基酸序列：YYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR4，其中所述H-FR4可包含SEQ ID NO:62所示的氨基酸序列：WGQGTX 6VX 8VSS，其中，X 6为L或S，且X 8为I或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR4可包含SEQ ID NO:28、40和41中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR4，其中所述H-FR4可包含SEQ ID NO:28所示的氨基酸序列：WGQGTLVTVSS。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含轻链可变区VH，其中所述VH可包含SEQ ID NO:55所示的氨基酸序列：X 1VQLQESGPGLVKPSX 16X 17LSLTCTVX 25GYSITSDYAWX 36WIRQX 41PGX 44X 45X 46EWX 49GYIX 53X 54SGX 57X 58X 59YNPSLKSRX 68X 69X 70X 71X 72DTSKNQFX 80LX 82LX 84X 85VTX 88X 89DTATYYCAX 98LX 100X 101X 102X 103YX 105X 106AMDYWGQGTX 116VX 118VSS，其中X 1为D或Q，X 16为E或Q，X 17为S或T，X 25为S或T，X 36为I或N，X 41为F或P，X 44为N或K，X 45为G、K或R，X 46为L或V，X 49为I或M，X 53为S或T，X 54为S或Y，X 57为A或S，X 58为P或T，X 59为R、S或Y，X 68为I或V，X 69为S或T，X 70为F或I，X 71为S或T，X 72为R或V，X 80为F或S，X 82为K或Q，X 84为S或T，X 85为F或S，X 88为A或T，X 89为A或E，X 98为R或S，X 100为D或G，X 101为F或T，X 102为D或G，X 103为N或Y，X 105为G或不存在，X 106为G或不存在，X 116为L或S，且X 118为I或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含重链可变区VH，其中所述VH可包含SEQ ID NO:1、9、12、14和32中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含轻链可变区VH，其中所述VH可包含SEQ ID NO: 54所示的氨基酸序列：QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX 44X 45LEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS，其中X 44为K或N，且，X 45为G或K。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含重链可变区VH，其中所述VH可包含SEQ ID NO:9、12和14中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含重链恒定区，所述重链恒定区可以来自人IgG，例如，人IgG1。在某些情形中，可以对人IgG1的Fc区进行修饰以达到所期望的性质(例如，ADCC KO)。所述修饰可以是氨基酸突变。在某些情形中，对IgG1的修饰可以为L234A/L235A，即，按照EU编号，第234位和第235位的氨基酸分别由亮氨酸(L)突变为丙氨酸(A)。在某些情形中，本申请所述的抗原结合蛋白的重链恒定区可以来自野生型的人IgG1。
例如，本申请所述的抗原结合蛋白可以包含重链恒定区，其可包含SEQ ID NO:35-36中任一项所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，其可以包含轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:64所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，其可以包含轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:63所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:2、10、11、13、33和34中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的VL可包含LCDR1、LCDR2和LCDR3。
本申请所述的抗原结合蛋白可以包含LCDR1，其中所述LCDR1可包含SEQ ID NO:65所示的氨基酸序列：QHVSX 5A，其中，X 5为N或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述LCDR1可包含SEQ ID NO:6和52中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含LCDR1，其中所述LCDR1可包含SEQ ID NO:6所示的氨基酸序列：QHVSTA。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含LCDR2，其中所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列：SAS。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含LCDR3，其中所述LCDR3可包含SEQ ID NO:66 所示的氨基酸序列：QQX 3YX 5X 6PX 8T，其中，X 3为H或Y，X 5为I或S，X 6为L或T，且X 8为W或Y。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述LCDR3可包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含LCDR3，其中所述LCDR3可包含SEQ ID NO:8所示的氨基酸序列：QQHYITPYT。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白还可以包含框架区L-FR1、L-FR2、L-FR3和L-FR4。
本申请所述的抗原结合蛋白可以包含框架区L-FR1，其中所述L-FR1可包含SEQ ID NO:68所示的氨基酸序列：DIX 3MTQSX 8X 9X 10X 11X 12X 13SX 15GDRVX 20ITCX 24AS；其中，X 3为Q或V，X 8为H或P，X 9为K或S，X 10为F或S，X 11为L或M，X 12为F或S，X 13为A或T，X 15为I或V，X 20为S或T，X 24为K或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR1可包含SEQ ID NO:15、16、44和45中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR1，其中所述L-FR1可包含SEQ ID NO:67所示的氨基酸序列：DIQMTQSPSSLSASVGDRVTITCX 24AS；其中，X 24为K或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR1可包含SEQ ID NO:15和16中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR2，其中所述L-FR2可包含SEQ ID NO:70所示的氨基酸序列：X 1X 2WYQQKPGX 10X 11PKLLIX 17；其中，X 1为L或V，X 2为A或N，X 10为K或Q，X 11为A或S，且，X 17为H或Y。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR2可包含SEQ ID NO:17、18、46和47中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR2，其中所述L-FR2可包含SEQ ID NO:69所示的氨基酸序列：X 1X 2WYQQKPGKAPKLLIY；其中，X 1为L或V，且X 2为A或N。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR2可包含SEQ ID NO:17和18中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR3，其中所述L-FR3可包含SEQ ID NO:72所示的氨基酸序列：YX 2X 3X 4GVPX8RFX 11GX 13X 14SGTDFTX 21TIX 24SX 26QX 28EDX 31AX 33YYC；其中，X 2为L或R，X 3为Q或Y，X 4为S或T，X 8为D或S，X 11为I、S或T，X 13为R或S，X 14为G或R，X 21为F或L，X 24为N或S，X 26为L或V，X 28为A或P，X 33为F或L，且X 14为T或V。例如，该序列可以是根据 IMGT定义规则确定的序列。
例如，所述L-FR3可包含SEQ ID NO:19-21和48-50中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR3，其中所述L-FR3可包含SEQ ID NO:71所示的氨基酸序列：YX 2X 3SGVPSRFSGSX 14SGTDFTLTISSLQPEDFATYYC；其中，X 2为L或R，X 3为Q或Y，且X 14为G或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR3可包含SEQ ID NO:19-21中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR4，其中所述L-FR4可包含SEQ ID NO:73所示的氨基酸序列：FGX 3GTKLEIK，其中，X 3为G或Q。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR4可包括SEQ ID NO:22和51中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR4，其中所述L-FR4可包含SEQ ID NO:22所示的氨基酸序列：FGQGTKLEIK。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:64所示的氨基酸序列：DIX 3MTQSX 8X 9X 10X 11X 12X1 3SX 15GDRVX 20ITCX 24ASQHVSX 31AX 33X 34WYQQKPGX 42X 43PKLLIX 49SASYX 54X 55X 56GVPX 60RFX 63GX 65X 66SGTDFTX 73TIX 76SX 78QX 80EDX 83AX 85YYCQQX 91YX 93X 94PX 96TFGX 100GTKLEIK，其中，X 3为Q或V，X 8为H或P，X 9为K或S，X 10为F或S，且，X 11为L或M，X 12为F或S，X 13为A或T，X 15为I或V，X 20为S或T，X 24为K或R，X 31为N或T，X 33为L或V，X 34为A或N，X 42为K或Q，X 43为A或S，X 49为H或Y，X 54为L或R，X 55为Q或Y，X 56为S或T，X 60为D或S，X 63为I、S或T，X 65为S或R，X 66为G或R，X 73为L或F，X 76为S或N，X 78为L或V，X 80为A或P，X 83为F或L，X 85为T或V，X 91为H或Y，X 93为I或S，X 94为L或T，X 96为W或Y，且，X 100为Q或G。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:2、10、11、13、33和34中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:63所示的氨基酸序列：DIQMTQSPSSLSASVGDRVTITCX 24ASQHVSTAX 33X 34WYQQKPGKAPKLLIYSASYX 54X 55 SGVPSRFSGSX 66SGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK，其中，X 24为K或R，X 33为L或V，X 34为A或N，X 54为L或R，X 55为Q或Y，且，X 66为G或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:10、11和13中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含轻链恒定区，所述轻链恒定区可以包含人轻链恒定区序列，例如，可以包含人κ轻链恒定区。例如，本申请所述分离的抗原结合蛋白的轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:4和42中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:5和43中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1、所述HCDR2和所述HCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1、所述HCDR2和所述HCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:42和SEQ ID NO:43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:65所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:66所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:6和52中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:6、SEQ  ID NO:7和SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:52、SEQ ID NO:7和SEQ ID NO:53所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:65所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:66所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:4和42中任一项所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:5和43中任一项所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:6和52中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、所述HCDR2、所述HCDR3、所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列。
在某些情形中，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、所述HCDR2、所述HCDR3、所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:7和SEQ ID NO:53所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:55所示的氨基酸序列，且所述VL可包含SEQ ID NO:64所示的氨基酸序列。
在某些情形中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:32和SEQ ID NO:14中任一项所示的氨基酸序列，且所述VL可包含SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:13中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:54所示的氨基酸序列，且所述VL可包含SEQ ID NO:63所示的氨基酸序列。
在某些情形中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9、SEQ ID NO:12和SEQ ID NO:14中任一项所示的氨基酸序列，且所述VL可包含SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:13中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:10所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:12所示的氨基酸序列，且所述VL可包含SEQ ID NO:13所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:14所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:1所示的氨基酸序列，且所述VL可包含SEQ ID NO:2所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:34所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:33所示的氨基酸序列。
本申请所述分离的抗原结合蛋白还可包含重链恒定区和轻链恒定区，所述重链恒定区可以来自人IgG1的恒定区，所述轻链恒定区可以来自人κ轻链恒定区。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:10所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为HB0030。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列； 且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列该分离的抗原结合蛋白可称为HB0031。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:12所示的氨基酸序列，且所述VL可包含SEQ ID NO:13所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为HB0032。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:14所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为HB0033。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:1所示的氨基酸序列，且所述VL可包含SEQ ID NO:2所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:30所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列该分离的抗原结合蛋白可称为900424。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:34所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:30所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为900423。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:33所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:30所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为900428。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、 HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:56所示的氨基酸序列；所述HCDR3可以包含SEQ ID NO:57所示的氨基酸序列，所述参比抗体的轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:65所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3可以包含SEQ ID NO:66所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:4所示的氨基酸序列；所述HCDR3可以包含SEQ ID NO:5所示的氨基酸序列，所述参比抗体的轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3可以包含SEQ ID NO:8所示的氨基酸序列。
在本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述抗原结合蛋白的氨基酸序列的一部分可以与来自特定物种的抗体中相应的氨基酸序列同源，或者属于特定的类别。例如，抗体的可变区及恒定部分均可以来自一个动物物种(如人)的抗体的可变区及恒定区。在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、 Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
本申请所述的抗原结合蛋白(例如，TIGIT抗体)能够特异性结合TIGIT抗原。“特异性结合”TIGIT抗原的抗原结合蛋白(例如，抗体)通常可以以约1nM的KD值或更高亲和力(例如，1nM、100pM、10pM、2pM或1pM)结合TIGIT，但不结合缺乏TIGIT序列的其它蛋白。例如，“特异性结合”TIGIT的抗体不结合人CD226、人CD155和人CD112。本申请所述的抗原结合蛋白(例如，抗体)能够特异性结合TIGIT抗原或其标记形式(例如，荧光标记的TIGIT抗原)，但不会结合缺乏TIGIT表位的其它蛋白。抗原结合蛋白(例如，抗体)是否结合TIGIT抗原可使用本领域中已知的任何测定法确定。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(例如，BIACORE)或类似技术(例如，KinExa或OCTET)。在某些情形中，本申请所述的TIGIT抗体还可以与猴和/或鼠的TIGIT交叉反应。例如，通过流式分析技术和酶联免疫反应所检测的。如本文所用，“交叉反应性”是指抗体与来自其它物种的同源蛋白反应的能力。
本申请所述的抗原结合蛋白(例如，TIGIT抗体)能够抑制TIGIT与CD155配体的结合。阻断实验可以使用竞争法进行检测，例如，将所述的抗原结合蛋白(例如，TIGIT抗体)与抗原(或，可表达抗原的细胞)和抗原的配体(或，表达配体的细胞)混合，根据可检测标记的的强度(例如，荧光强度或浓度)反应抗原结合蛋白与抗原的配体竞争性结合抗原的能力。例如，如使用流式细胞仪所检测的，本申请所述的抗原结合蛋白(例如，TIGIT抗体)阻断TIGIT抗原与CD155配体结合的IC50为约0.1μg/ml-0.05μg/ml。
核酸、载体和细胞
在另一个方面，本申请还提供了分离的一种或多种核酸分子。所述一种或多种核酸分子可编码本申请所述的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体、其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.，1993。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种 核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
制备方法
在另一个方面，本申请提供了制备所述的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白(例如，抗TIGIT抗体)。例如，可以用连接或天然存在的TIGIT同源二聚体或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的TIGIT都可以作为免疫原(抗原)，用于产生对TIGIT特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人TIGIT，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、腺相关病毒载体、杆状病毒载体、质料和非病毒载体。
生产本申请的抗人TIGIT抗体的示例性方法描述于实施例1。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体是IgG抗体，使用IgG1亚型。可以通过用下文实施例中描述的生物学测定筛选抗体实现必需恒定结构域序列的优化，以产生所需生物学活性。同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本申请的化合物和方法中是可以用的。
人源化本申请的抗人TIGIT抗体的示例性方法描述于实施例2。
本申请的抗原结合蛋白或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该核酸分子引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请还涉及包含上述的适当核酸分子以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。例如，动物细胞可以包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
本申请中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本申请的核酸分子所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。通常，在适合本申请抗原结合蛋白表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的抗原结合蛋白。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如流式细胞分选技术(FACS)、放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
药物组合物
另一方面，本申请还提供了一种组合物。在某些情形中，所述的组合物可以是药物组合物，它含有本申请的抗原结合蛋白或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常可以为约5-8，例如，pH可以为约6-8，pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本申请所述的抗原结合蛋白也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本申请所述的药物组合物可直接用于结合TIGIT蛋白分子，因而可用于预防和治疗TIGIT相关的疾病。此外，还可同时使用其他治疗剂。
本申请的药物组合物可以含有安全有效量(如0.001-99wt％，0.01-90wt％，或0.1-80wt％) 的本申请所述的抗原结合蛋白以及药学上可接受的佐剂(可包括载体或赋形剂)。这类载体可以包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本申请所述的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重至约5毫克/千克体重。此外，本申请所述的抗原结合蛋白还可与其他治疗剂一起使用。
本文所述的抗原结合蛋白或药物组合物可以符合良好医疗实践的方式配制、给药和施用。在此情形下的考虑因素包括所治疗的特定病症、所治疗的特定哺乳动物、单个患者的临床病状、病症的病因、药剂递送部位、施用方法、施用排程和医学从业者已知的其他因素。治疗剂(例如，抗TIGIT抗体)无需但任选地与一种或多种当前用来预防或治疗所考虑的病症的药剂一起配制和/或同时施用。此类其他药剂的有效量取决于制剂中存在的治疗剂(例如，抗TIGIT抗体)的量、病症或治疗的类型以及以上论述的其他因素。这些药剂通常可以凭经验/临床上确定为适当的任何剂量且通过凭经验/临床上确定为适当的任何途径加以使用。与单个治疗相比，可减少组合治疗中施用的抗体的剂量。通过常规技术易于监测此疗法的进展。
方法和用途
另一方面，本申请提供了所述抗原结合蛋白、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备药物中的用途。所述药物用于预防、缓解和/或治疗TIGIT相关的疾病。在某些情形中，所述TIGIT相关的疾病可以是T细胞功能障碍性病症。T细胞功能障碍体现在T细胞耗尽，是通过增强NK细胞和激活T细胞，增强机体免疫活性实现对疾病的治疗或延迟或缓解。例如，所述TIGIT相关的疾病可以是肿瘤、癌症或感染性病症。例如，所述TIGIT相关的疾病可以是CD155阳性或PVR阳性的肿瘤、癌症、免疫性疾病或感染性病症，包括肿瘤、癌症、免疫性疾病或感染性病症等。在某些情形中，所述肿瘤可以选自下组：乳腺癌、结肠癌、肝癌、淋巴瘤、软骨肉瘤、多发性骨髓瘤、肺癌、肾癌、黑色素瘤、T淋巴瘤、胰腺癌。例如，所述肿瘤为结肠癌。例如，本申请所述的抗原结合蛋白可以抑制选自下组的肿瘤动物模型的肿瘤生长：乳腺癌、结肠癌、肝癌、淋巴瘤、软骨肉瘤、多发性骨髓瘤、肺癌、肾癌、黑色素瘤、T淋巴瘤和胰腺癌。
本申请的抗原结合蛋白可以抑制肿瘤生长和/或抑制肿瘤细胞增殖。在某些实施方式中，所述肿瘤包含结肠癌。在某些实施方式中，所述肿瘤或癌症为TIGIT表达异常的肿瘤或癌症。本申请还提供了以下描述的检测生物样品中TIGIT表达的方法。在某些情形中，所述方法包括使生物样品与本申请所述的抗原结合蛋白在容许所述抗原结合蛋白结合TIGIT的条件下接 触，和检测在所述抗原结合蛋白与TIGIT之间是否形成复合物。例如，所述肿瘤或癌症为与非肿瘤或癌症样品相比，TIGIT表达升高的肿瘤或癌症。此类方法可以是体外或体内方法。本申请所述抗原结合蛋白可用于例如免疫测定中，所述免疫测定包括例如免疫组织化学(IHC)、免疫荧光(IF)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FAGS)和酶联免疫吸附测定(ELISA)。在某些情形中，例如当TIGIT为用于选择患者的生物标记时，所述抗原结合蛋白用来选择适于用本申请所述抗原结合蛋白和/或融合蛋白进行的疗法的受试者。本申请还提供了所述抗原结合蛋白在诊断患有病症(例如，癌症或免疫功能失调)的受试者的方法中的用途，所述方法包括：通过使样品与本申请的所述抗原结合蛋白接触并检测结合的所述抗原结合蛋白和/或融合蛋白的存在来确定获自受试者的样品中TIGIT的存在或表达水平。
例如，在结肠癌小鼠模型中，本申请所述的抗原结合蛋白(例如，TIGIT抗体)能够减缓肿瘤生长。
本申请的抗原结合蛋白可用于检测应用，例如用于检测样本，从而提供诊断信息。
在本申请中，所采用的样本(样品)包括细胞、组织样本和活检标本。本申请使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本申请中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本申请中使用的样本包括固定的或保存的细胞或组织样本。
本申请还提供了一种指含有本申请的抗原结合蛋白的试剂盒。在某些情形中，所述的试剂盒还可以包括容器、使用说明书、缓冲剂等。例如，本申请的原结合蛋白可以固定于检测板。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、制备方法和用途等，而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例
实施例1 抗人TIGIT的小鼠单克隆抗体——原始抗体的制备方法
1.1制备产生鼠源单克隆抗体的杂交瘤细胞
制备鼠源单克隆抗体的方法采用Kohler和Milstein 1975年发明的杂交瘤制备技术(Nature，1975，256：495-497)。首先将人TIGIT带有鼠Fc标签蛋白(ACRO，#TIT-H5253)与弗氏佐 剂乳化，然后对BALB/c、CD1、C57BL/6每个品系各5只小鼠进行多点皮下免疫。三轮免疫后取血清用ELISA法检测效价，FACS检测结合活性及功能活性，挑选最佳小鼠取脾细胞与SP2/0骨髓瘤细胞进行融合。经过HAT筛选杂交瘤多克隆细胞，采用ELISA、FACS方法，筛选出特异性结合人TIGIT且可以阻断TIGIT-CD155结合的多克隆细胞株后进行单克隆化，再次使用ELISA、FACS方法筛选特异性结合的单克隆细胞株，并检测了与猴、小鼠TIGIT结合力，通过FACS筛选出阻断TIGIT-CD155与TIGIT-CD122结合的单克隆抗体，后续进行细胞功能学活性检测，并对筛选出的单克隆细胞株进行亲和力(Biacore)筛选，最终得到表达人TIGIT抗体的单克隆杂交瘤细胞株进行序列分析，筛选数据列举见表1。
表1.杂交瘤筛选数据
Figure PCTCN2021092043-appb-000001
经过高通量筛选，最终得到的单克隆杂交瘤细胞株具有高亲和力，可以与人和猴都具有结合活性，对TIGIT的两个配体CD155及CD122同时具有阻断功能，并且具有生物活性。 将以上表1中的杂交瘤细胞株进行人源化改造。
实施例2 抗TIGIT抗体可变区基因序列克隆以及人源化
2.1 杂交瘤细胞中抗体的可变区基因克隆
基于TAKARA的5’RACE技术原理，克隆出由杂交瘤细胞株表达的小鼠抗体可变区的cDNA序列。简言之，用SMARTer 5’RACE合成试剂盒(TAKARA，#634859)按说明书合成重链和轻链的可变区基因特异性cDNA。用PCR引物修饰cDNA序列的5’和3’端，所述引物设计成为分别在重链和轻链可变区cDNA上增加合适的前导序列，使所得PCR产物能够通过无缝克隆的方法克隆到现有重组抗体表达的重链载体pHB-Fc和轻链载体pHB-Cκ上。pHB-Fc表达载体上含有人IgG1重链恒定区基因序列，其中CH 2上带有抗体ADCC knock out(KO)效应的L234A和L235A(Eu编码)突变；pHB-Cκ载体上含有人κ轻链恒定区基因序列。将重链和轻链可变区PCR扩增产物通过In-fusion克隆试剂(TAKARA,#639650)克隆到表达载体上，并转化到E.coli DH5α大肠杆菌感受态细胞(益生生技，#FYE607-80VL)中。通过挑选单克隆菌落进行Sanger测序，经分析获得抗体可变区序列。其中B3/29F6表达的抗TIGIT抗体可变区序列如下：
B3/29F6 VH    SEQ ID NO:1
Figure PCTCN2021092043-appb-000002
B3/29F6 VL    SEQ ID NO:2
Figure PCTCN2021092043-appb-000003
其中，下划线区为CDRs(按照IMGT定义，序列分别如下)：
表2.鼠源抗TIGIT抗体CDR序列
Figure PCTCN2021092043-appb-000004
2.2 IgG1野生型嵌合表达载体构建
IgG1野生型为嵌合抗体和ADCC KO型嵌合抗体共用相同的轻链载体，不同之处在于所用含有人重链恒定区序列的载体上重链CH 2的234和235位氨基酸，ADCC KO型为A234/A235，野生型为L234/L235。IgG1野生型嵌合重链表达载体的方法与2.1中所述表达载体构建方法原理相同，即通过含有前导序列的引物PCR扩增重链可变区基因，再用无缝连接方法克隆到含有人重链恒定区序列(CH2为L234/L235)的载体上，完成IgG1野生型嵌合表达载体的构建。
2.3 嵌合抗体的表达
2.1和2.2中所得表达载体经过大肠杆菌扩增，用去内毒素质粒抽提试剂盒(天根生化科技(北京)有限公司，#DP117)制备足量质粒，用于瞬时转染表达嵌合抗体。表达所用的宿主细胞为CHO-S细胞(赛默飞，#R80007)。通过将制备所得的两种重链载体分别和轻链载体一起，与聚醚酰亚胺(PEI，Polysciences，#24765-1)混合形成脂质体复合物后，转染CHO-S细胞，放入培养箱中培养5-7天。离心收集细胞培养液上清，通过Protein A亲和层析柱纯化得到ADCC KO型人鼠嵌合抗体(编号为900424)和IgG1野生型人鼠嵌合抗体(蛋白编号900445)。
按照上述方法得到的嵌合抗体VH和VH序列分别如下：
900423和900428 VH    SEQ ID NO:32
Figure PCTCN2021092043-appb-000005
900424 VH    SEQ ID NO:1
Figure PCTCN2021092043-appb-000006
900424 VL    SEQ ID NO:2
Figure PCTCN2021092043-appb-000007
900423 VL    SEQ ID NO:34
Figure PCTCN2021092043-appb-000008
900428 VL    SEQ ID NO:33
Figure PCTCN2021092043-appb-000009
Figure PCTCN2021092043-appb-000010
其中，900424的重链恒定区氨基酸序列为SEQ ID NO:30所示，900423的重链恒定区氨基酸序列为SEQ ID NO:30所示，900428的重链恒定区氨基酸为SEQ ID NO:30所示。轻链恒定区均为人κ轻链恒定区(SEQ ID NO:31)。
2.4 鼠源抗人TIGIT抗体的人源化——人源化抗体的制备方法
抗体的人源化采用以下方法。将抗体的可变区序列与NCBI IgBlast数据库中的可用序列比较，通过鉴定和分析，最终确定了适合在其上构建CDR移植重链和轻链的人源构架区(FR区)。
改造时，根据人抗体FR区保守的氨基酸残基以及抗体FR区中重要的氨基酸残基，设计改造位点，对嵌合抗体的重轻链的可变区分别进行人源化突变设计，利用PCR技术扩增并构建人源化点突变抗体表达质粒。将人源化点突变抗体表达质粒分别经CHO-S细胞表达，纯化后得到人源化抗体蛋白。利用ELISA，Biacore和流式细胞术等检测方法，对人源化抗体的受体结合能力，功能抑制活性和ADCC效应等指标进行筛选，获得了四个性能优异的人源化抗TIGIT抗体。所获得人源化抗TIGIT抗体的VH和VL序列下所示：
900461和900464 VH    SEQ ID NO:9
Figure PCTCN2021092043-appb-000011
900461 VL    SEQ ID NO:10
Figure PCTCN2021092043-appb-000012
900464和900476 VL    SEQ ID NO:11
Figure PCTCN2021092043-appb-000013
900466 VH    SEQ ID NO:12
Figure PCTCN2021092043-appb-000014
900466 VL    SEQ ID NO:13
Figure PCTCN2021092043-appb-000015
Figure PCTCN2021092043-appb-000016
900476 VH    SEQ ID NO:14
Figure PCTCN2021092043-appb-000017
其中，下划线区为CDRs(按照IMGT定义)，900461，900464，900466和900476分别为四个人源化抗体蛋白编号，对应项目编号为HB0030、HB0031、HB0032、HB0033。
其中，HB0030的重链恒定区氨基酸序列为SEQ ID NO:29所示，HB0031的重链恒定区氨基酸序列为SEQ ID NO:29所示，HB0032的重链恒定区氨基酸序列为SEQ ID NO:29所示，HB0033的重链恒定区氨基酸序列为SEQ ID NO:29所示。轻链恒定区均为人κ轻链恒定区(SEQ ID NO:31)。
实施例3 嵌合抗体及人源化抗体的检测
3.1 测试TIGIT抗体对表达人TIGIT的细胞结合活性
嵌合抗体900324、900423、900424、900428，人源化抗体HB0030、HB0031、HB0032、HB0033与表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)的结合活性检测。
所有嵌合抗体、人源化抗体均用含1％BSA的PBS溶液(1％BSA/PBS)稀释至20μg/ml，每孔20μL加入96孔U型板中，同步设定阴性对照(只加1％BSA/PBS)。取对数生长期内表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)悬液，离心(1000rpm×5min)弃培养液，用1％BSA/PBS重悬至活细胞密度为1×10 6/mL，每孔20μL(2×10 4个细胞)加入已有抗TIGIT抗体的96孔U型板中室温反应30min。将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤1遍，加入1:200稀释的PE-羊抗人-Fc(Jackson ImmunoResearch，#109-115-098)，室温避光反应15min；将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤3遍，最终用每孔100μL 1％BSA/PBS重悬，用流式细胞仪(BD，#CantoⅡ)检测PE通道的荧光强度。
嵌合抗体结合活力结果见图1及表3，人源化抗体结合活力结果见图2及表4，其中900324为CHO细胞瞬时表达的来自基因泰克公司的抗人TIGIT抗体Tiragolumab，其轻重链可变区序列与专利申请WO2015009856A2中的抗体10A7的序列相同。结果表明嵌合抗体与人源化抗体均与表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)有很好的亲和力，且亲和力相当。
表3.抗TIGIT嵌合抗体对人TIGIT结合活性
抗体 900428 900424 900423 900324
EC 50(μg/mL) 0.075 0.053 0.06 0.124
表4.抗TIGIT人源化抗体对人TIGIT结合活性
抗体 900324 HB0030 HB0031 HB0032 HB0033
EC 50(μg/mL) 0.08994 0.04661 0.05806 0.07847 0.0514
3.2 测试TIGIT抗体阻断TIGIT抗原和TIGIT分子结合的实验(竞争法)
将抗原huCD155-moFc(ACROBiosystems，#CD5-H5254)用含1％BSA的PBS溶液(1％BSA/PBS)稀释至40μg/ml，每孔10μL加入96孔U型板中，与系列稀释的抗TIGIT抗体按体积比1:1混合均匀，同步设定阳性对照(只加CD155-moFc)。取对数生长期内表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)悬液，离心(1000rpm×5min)弃培养液，用1％BSA/PBS重悬至活细胞密度为1×10 6/mL，每孔20μL(2×10 4个细胞)加入CD155-moFc与抗TIGIT抗体混匀的96孔U型板中室温反应30min。将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤1遍，加入1:300稀释的Alexa488-羊抗鼠-Fc(Jackson ImmunoResearch，#115-545-071)，室温避光反应15min；将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤3遍，最终用每孔100μL1％BSA/PBS重悬，用流式细胞仪(BD，#CantoⅡ)检测FITC通道的荧光强度。
嵌合抗体结合活力结果见图3及表5，人源化抗体结合活力结果见图4及表6。结果表明嵌合抗体和人源化抗体对人的CD155具有显著的阻断作用，且阻断作用力相当。
表5.抗TIGIT嵌合抗体对人TIGIT与其配体CD155结合的阻断活性
抗体 900428 900424 900423 900324
IC 50(μg/mL) 0.069 0.062 0.065 0.174
表6.抗TIGIT人源化抗体对人TIGIT与其配体CD155结合的阻断活性
抗体 900324 HB0030 HB0031 HB0032 HB0033
IC 50(μg/mL) 0.1349 0.05733 0.06653 0.0919 0.05598
3.3 人源化单克隆抗体的亲和性检测
本试验使用SPR方法测定抗原-抗体结合动力学及亲和力。采用BIOCORE(GE，#Biacore8K)。Sereis S Sensor Chip ProteinA芯片(GE，#29-1275-56)室温平衡20～30min，将芯片装入仪器。用平衡缓冲液将抗体样品稀释至实验工作浓度，2～8℃密封备用。用平衡缓冲液HBS-EP(10x)(GE，#BR-1006-69)稀释抗原，组氨酸标记(His-Tag)的人TIGIT蛋白(huTIGIT，ACRO Biosystems，#TIT-H52H3)，稀释抗原20nM起始2.5倍稀释度5个浓度梯度，并设置2个零浓度(即平衡缓冲液)和最低浓度重复。pH1.5甘氨酸溶液(GE，#BR100354)再生芯 片。采用捕获法多循环动力学程序分析样品，选用对应的分析程序对数据分析，确认无明显reference binding，选用Kinetics，1:1binding modle，拟合分析，获得样品的动力学参数。
检测结果如表7所示。与huTIGIT的亲和力常数(KD(M))结果显示，本申请的人源化单克隆抗体HB0030的亲和力接近10 -11数量级，具有极强的亲和力。
表7.人源化Mab-TIGIT抗体与hu TIGIT亲和力检测结果
抗体 Ka(1/Ms) Kd(1/s) KD(M)
HB0030 3.17E+06 1.85E-04 5.83E-11
实施例4 动物体内药效试验
本实验研究了抗人TIGIT抗体HB0030、HB0031、HB0032、HB0033在免疫检测点人源化小鼠BALB/c-hPD1/hTIGIT皮下接种CT26.WT结肠癌肿瘤模型中的药效。
本实验取用小鼠结肠癌细胞CT26.WT细胞，收集对数生长期的CT26.WT细胞，去除培养液并用PBS洗两次后接种(荷瘤前、荷瘤后CT26.WT细胞存活率分别为：98.57％及98.28％)，接种量：5×10 5/100μL/只。接种后第11天，平均肿瘤体积达到94.02mm 3时，小鼠根据肿瘤体积随机分组，每组8只。分组当天定义为D0天，并于D0、D3、D7、D10、D14、D17进行给药，开始给药后，于D0、D2、D4、D6、D8、D10、D13、D15、D17、D20、D23观测肿瘤大小并称量小鼠体重。瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。结果如图5所示。
观察图5实验结果，可见本申请的抗TIGIT人源化单克隆抗体HB0030、HB0031、HB0032、HB0033在单独给药10mg/kg时具有明显的肿瘤抑制效果。
进一步的研究了HB0030与对照抗体Tiragolumab在10mg/kg的剂量下，对免疫检测点人源化小鼠BALB/c-hPD1/hTIGIT皮下接种CT26.WT结肠癌肿瘤模型中的药效。
本实验取用小鼠结肠癌细胞CT26细胞，收集对数生长期的CT26细胞，去除培养液并用PBS洗两次后接种(荷瘤前、荷瘤后CT26细胞存活率分别为：98.51％及97.250％)，接种量：5×10 5/100μL/只，接种第12天，平均肿瘤体积达到80.33mm 3时，小鼠根据肿瘤体积随机分组，每组6只。分组当天定义为D0天，并于D0、D3、D7、D10、D14、D17进行给药，开始给药后，于D0、D2、D4、D6、D8、D10、D13、D15、D17、D20、D23观测肿瘤大小并称量小鼠体重。瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。结果如图6所示。
观察图6结果可知，本申请的抗TIGIT抗体HB0030与对照抗体Tiragolumab比较，表现出更好的肿瘤抑制效果。
本研究中所有组小鼠治在接种后24天，小鼠体重均无明显变化。
实施例5 抗TIGIT抗体HB0030在动物体内药效实验
为评估HB0030分子ADCC作用在体内药效的作用，其中900541为Fc区经过L234A/L235A突变的不具有ADCC作用的HB0030，另外两个为HB0030不同批次的样品。本实验以生理盐水为阴性对照评价了以上三个药物在小鼠结肠癌细胞CT26.WT移植人源化小鼠BALB/c-hPD1/hTIGIT中的抗肿瘤作用。
BALB/c-hPD1/hTIGIT小鼠(接种CT26.WT细胞)分别腹腔注射3mg/kg测试药,每周2次，共给药6次。与对照组G1(生理盐水)相比：根据肿瘤体积结果(TIGtv)和体重结果(TIGtw)分析，900541(G2:anti-TIGIT-1，3mpk)抑瘤率分别为TGItv 40.26％、TGItw 32.60％；抗TIGIT抗体HB0030(G3:anti-TIGIT-2，3mpk)抑瘤率分别为TGItv 90.68％、TGItw 86.85％，对肿瘤生长具有显著抑制作用(G3VS G1，***Ptv<0.001,***Ptw<0.001)；抗TIGIT抗体HB0030(G4:anti-TIGIT-3，3mpk)抑瘤率分别为TGItv 82.38％、TGItw 77.85％，对肿瘤生长具有显著抑制作用(G4VS G1，***Ptv<0.001,***Ptw<0.001)。此外，(G3:anti-TIGIT-2，3mpk)与HB0030(G4:anti-TIGIT-3，3mpk)之间肿瘤抑制作用无明显差别。(G4VS G2，Ptv>0.263,Ptv>0.346)
综上所述，在当前的测试系统下，BALB/c-hPD1/hTIGIT小鼠(接种CT26.WT细胞)分别腹腔注射3mg/kg测试药,每周2次，共给药6次，与生理盐水对照组相比，G2的900541、G3和G4的HB0030(3mpk)均表现出明显的肿瘤抑制作用。

Claims (67)

  1. 分离的抗原结合蛋白，其包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:55所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:64所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    1)能够以1×10 -10M或更低的KD值结合TIGIT蛋白，其中所述KD值通过表面等离子体共振法测定；
    2)在FACS测定中，能够阻断CD155与TIGIT的结合；和
    3)能够抑制肿瘤生长和/或肿瘤细胞增殖。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体包含重链可变区和轻链可变区，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列；
    所述HCDR2包含SEQ ID NO:56所示的氨基酸序列；所述HCDR3包含SEQ ID NO:57所示的氨基酸序列，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:65所示的氨基酸序列；所述LCDR2包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3包含SEQ ID NO:66所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  6. 根据权利要求5所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab)2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。
  7. 根据权利要求5-7中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包含HCDR1，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包含HCDR2，所述HCDR2包含SEQ ID NO:56所示的氨基酸序列。
  10. 根据权利要求9所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:4和42中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包含HCDR3，所述HCDR3包含SEQ ID NO:57所示的氨基酸序列。
  12. 根据权利要求11所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:5和43 所示的氨基酸序列。
  13. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，且所述分离的抗原结合蛋白包含选自下述的任意一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:3，HCDR2：SEQ ID NO:4，和HCDR3：SEQ ID NO:5；以及
    (2)HCDR1：SEQ ID NO:3，HCDR2：SEQ ID NO:42，合HCDR3：SEQ ID NO:43。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包含VL，其中所述VL包含LCDR1，所述LCDR1包含SEQ ID NO:65所示的氨基酸序列。
  15. 根据权利要求14所述的分离的抗原结合蛋白，所述LCDR1包含SEQ ID NO:6和52中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含VL，其中所述VL包含LCDR2，所述LCDR2包含SEQ ID NO:7所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含VL，其中所述VL包含LCDR3，所述LCDR3包含SEQ ID NO:66所示的氨基酸序列。
  18. 根据权利要求17所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，且所述分离的抗原结合蛋白包含选自下述的任意一组氨基酸序列：
    (1)LCDR1：SEQ ID NO:6，LCDR2：SEQ ID NO:7，和，LCDR3：SEQ ID NO:8；以及
    (2)LCDR1：SEQ ID NO:52，LCDR2：SEQ ID NO:7，和，LCDR3：SEQ ID NO:53。
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包含VL，且所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:68所示的氨基酸序列。
  21. 根据权利要求20所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:15、16、44和45中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包含VL，且所述VL包括框架区L-FR2，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:70所示的氨基酸序列。
  23. 根据权利要求22所述的分离的抗原结合蛋白，其中所述L-FR2包含SEQ ID NO:17、18、46和47中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含VL，且所述VL包括框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:72所示的氨基酸序列。
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:19、20、21、48、49和50中任一项所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的分离的抗原结合蛋白，其包含VL，且所述VL包括框架区L-FR4，其中所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:73所示的氨基酸序列。
  27. 跟据权利要求26所述的分离的抗原结合蛋白，其中所述L-FR4包含SEQ ID NO:22和51中任一项所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包含VL，其中所述VL包含SEQ ID NO:64所示的氨基酸序列。
  29. 根据权利要求28所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:2、10、11、13、33和34中任一项所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻链恒定区来自人κ轻链恒定区。
  31. 根据权利要求30所述的分离的抗原结合蛋白，其中所述人κ轻链恒定区包含SEQ ID NO:31所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:58所示的氨基酸序列。
  33. 根据权利要求32所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:23和35中任一项所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包括框架区H-FR2，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:60所示的氨基酸序列。
  35. 根据权利要求34所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:24-26、36和37中任一项所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包括框架区H-FR3，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包 含SEQ ID NO:61所示的氨基酸序列。
  37. 根据权利要求36所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:27、38和39中任一项所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包括框架区H-FR4，其中所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:62所示的氨基酸序列。
  39. 根据权利要求38所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:28、40和41中任一项所示的氨基酸序列。
  40. 根据权利要求1-39中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包含SEQ ID NO:55所示的氨基酸序列。
  41. 根据权利要求40所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:1、9、12、14和32中任一项所示的氨基酸序列。
  42. 根据权利要求1-41中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
  43. 根据权利要求1-42中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区包括人IgG1恒定区，且人IgG1恒定区包含SEQ ID NO:29-30中任一项所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH和轻链可变区VL，且所述分离的抗原结合蛋白包含选自下述的任意一组氨基酸序列：
    (1)VH：SEQ ID NO:9，和VL：SEQ ID NO:10，
    (2)VH：SEQ ID NO:9，和VL：SEQ ID NO:11，
    (3)VH：SEQ ID NO:12，和VL：SEQ ID NO:13，
    (4)VH：SEQ ID NO:14，和VL：SEQ ID NO:11，
    (5)VH：SEQ ID NO:1，和VL：SEQ ID NO:2，
    (6)VH：SEQ ID NO:32，和VL：SEQ ID NO:34，以及
    (7)VH：SEQ ID NO:32，和VL：SEQ ID NO:33。
  45. 分离的一种或多种核酸分子，其编码权利要求1-44中任一项所述的分离的抗原结合蛋白。
  46. 载体，其包含根据权利要求45所述的核酸分子。
  47. 细胞，其包含根据权利要求45所述的核酸分子或根据权利要求46所述的载体。
  48. 多肽，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白。
  49. 免疫缀合物，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白或权利要求48所 述的多肽。
  50. 制备权利要求1-44中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-44中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求47所述的细胞。
  51. 药物组合物，其包含权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45所述的核酸分子、权利要求46所述的载体、权利要求47所述的细胞、权利要求48所述的多肽和/或权利要求49所述的免疫缀合物，以及任选地药学上可接受的佐剂。
  52. 权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45所述的核酸分子、权利要求46所述的载体、权利要求47所述的细胞、权利要求48所述的多肽、权利要求49所述的免疫缀合物和/或权利要求51所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗TIGIT相关疾病。
  53. 根据权利要求52所述的用途，所述TIGIT相关疾病为T细胞功能障碍性病症。
  54. 根据权利要求52-53中任一项所述的用途，所述TIGIT相关疾病为肿瘤。
  55. 根据权利要求54所述的用途，所述肿瘤为实体瘤。
  56. 根据权利要求54-55中任一项所述的用途，所述肿瘤为结肠癌。
  57. 抑制CD155与TIGIT结合的方法，所述方法包括施用权利要求1-44中任一项所述的分离的抗原结合蛋白。
  58. 预防、缓解或治疗TIGIT相关疾病的方法，所述方法包括向有需要的受试者施用权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45所述的核酸分子、权利要求46所述的载体、权利要求47所述的细胞、权利要求48所述的多肽、权利要求49所述的免疫缀合物和/或权利要求51所述的药物组合物。
  59. 根据权利要求58所述的方法，所述TIGIT相关疾病为T细胞功能障碍性病症。
  60. 根据权利要求58-59中任一项所述的方法，所述TIGIT相关疾病为肿瘤。
  61. 根据权利要求60所述的方法，所述肿瘤为实体瘤。
  62. 根据权利要求60-61中任一项所述的方法，所述肿瘤为结肠癌。
  63. 权利要求1-44中任一项所述的分离的抗原结合蛋白、权利要求45所述的核酸分子、权利要求46所述的载体、权利要求47所述的细胞、权利要求48所述的多肽、权利要求49所述的免疫缀合物和/或权利要求51所述的药物组合物，其用于预防、缓解或治疗TIGIT相关疾病。
  64. 根据权利要求63所述的分离的抗原结合蛋白、核酸分子、载体、细胞、多肽、免疫缀合 物和/或药物组合物，所述TIGIT相关疾病为T细胞功能障碍性病症。
  65. 根据权利要求64所述的分离的抗原结合蛋白、核酸分子、载体、细胞、多肽、免疫缀合物和/或药物组合物，所述TIGIT相关疾病为肿瘤。
  66. 根据权利要求64-65所述的分离的抗原结合蛋白、核酸分子、载体、细胞、多肽、免疫缀合物和/或药物组合物，所述肿瘤为实体瘤。
  67. 根据权利要求66所述的分离的抗原结合蛋白、核酸分子、载体、细胞、多肽、免疫缀合物和/或药物组合物，所述肿瘤为结肠癌。
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