WO2019062832A1 - Tigit抗体、其抗原结合片段及医药用途 - Google Patents

Tigit抗体、其抗原结合片段及医药用途 Download PDF

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WO2019062832A1
WO2019062832A1 PCT/CN2018/108246 CN2018108246W WO2019062832A1 WO 2019062832 A1 WO2019062832 A1 WO 2019062832A1 CN 2018108246 W CN2018108246 W CN 2018108246W WO 2019062832 A1 WO2019062832 A1 WO 2019062832A1
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seq
variable region
chain variable
antibody
antigen
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PCT/CN2018/108246
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French (fr)
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付雅媛
曹卓晓
胡齐悦
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to US16/651,764 priority Critical patent/US11512129B2/en
Priority to CN201880058441.3A priority patent/CN111051347B/zh
Priority to JP2020517291A priority patent/JP7317272B2/ja
Priority to EP18863534.6A priority patent/EP3689909A4/en
Publication of WO2019062832A1 publication Critical patent/WO2019062832A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • C07K2317/56—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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • 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

  • the present disclosure relates to TIGIT antibodies and antigen-binding fragments thereof. Further, the present disclosure also relates to chimeric antibodies, humanized antibodies comprising the CDR regions of the TIGIT antibodies, and to the inclusion of the TIGIT antibodies and antigen-binding fragments thereof. Pharmaceutical compositions, and their use as diagnostic and therapeutic agents for TIGIT related diseases.
  • T cells are key mediators of immune responses, and T cell activation is dependent on TCR signaling and costimulatory signals.
  • the costimulatory signal is a limiting signal for T cell activation, and its dysfunction is involved in the development of autoimmune diseases (Immunol Rev, 2012, 248: 122-139; Autoimmun Rev, 2013, 12: 1171-1176).
  • TIGIT T cell immunoglobulin and ITIM domain
  • the TIGIT gene is located on human chromosome 16, encoding a type I transmembrane protein consisting of 244 amino acids.
  • the human extracellular domain of TIGIT has a length of 141 amino acids and one immunoglobulin V-like domain; the transmembrane region has 23 amino acids; the cytoplasmic region is short, with 80 amino acids, and has a PDZ binding domain and ITIM motifs.
  • the TIGIT molecule belongs to the immunoglobulin superfamily IgSF, its structure is relatively conservative, and its homologous molecules are found in many mammals.
  • the human TIGIT molecule has 88% and 67% of TIGIT molecules in monkeys, dogs and mice, respectively. % and 58% homology (Nat Immunol, 2009, 10(1): 48-57).
  • TIGIT molecules are mainly expressed on the surface of T cells and NK cells (Nat Immunol, 2009, 10: 48-57). Both T cells and resting memory T cells underexpress TIGIT and up-regulate expression after in vitro activation (J Immunol, 2012, 188: 3869-3875). TIGIT on the surface of NK cells has a higher level of expression (Proc Natl Acad Sci USA, 2009, 106(42): 17858-17863). TIGIT is a potential new target for immunotherapy. Studies have shown that monoclonal antibodies that specifically block TIGIT show significant anti-tumor effects in animal models (Martinet and Smyth 2015).
  • TIGIT antibody promotes the killing function of CD8 T cells against HIV and melanoma, and this effect disappears with the blockade of CD226 (Chew, Fujita et al. 2016).
  • CD226 blockade of CD226
  • monoclonal antibody drug that blocks TIGIT at home and abroad, so it is not necessary to develop a highly specific TIGIT monoclonal antibody.
  • TIGIT antibodies and related applications of TIGIT have been reported in patents such as WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2017053748, WO2017030823, WO2016106302, US20160176963, US20130251720.
  • WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2017053748, WO2017030823, WO2016106302, US20160176963, US20130251720 no TIGIT antibody has been used in clinical practice, and new TIGIT antibodies that are more suitable for clinical applications still need to be developed.
  • the present disclosure provides monoclonal antibodies or antigen-binding fragments (also referred to as TIGIT binding molecules) that specifically bind to the amino acid sequence or three-dimensional structure of the extracellular region of TIGIT.
  • a monoclonal antibody or antigen-binding fragment thereof is provided, the monoclonal antibody or antigen-binding fragment specifically binding to a human TIGIT, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group consisting of the amino acid sequences of SEQ ID NOs: 15, 16 and 17, or HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 15, 16 and 17, respectively
  • An HCDR variant having a 3, 2 or 1 amino acid difference the light chain variable region comprising an LCDR1, LCDR2 and LCDR3 selected from the group consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20 or with SEQ ID NO: 18, 19 And LCDR1, LCDR1 and LCDR3 shown in 20, respectively, having an LCDR variant of 3, 2 or 1 amino acid difference; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the amino acid sequences of SEQ ID NO: 21-23 or HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 21-23, respectively, 3, 2 Or an 1 amino acid difference HCDR variant comprising a light crystal chain variable region selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 24-26 or with LCDR1, LCDR2 as shown in SEQ ID NOs: 24-26 LCDR variant with 3, 2 or 1 amino acid difference from LCDR3; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the amino acid sequences of SEQ ID NOs: 27-29 or HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 27-29, respectively, 3, 2 Or an amino acid variant HCDR variant comprising a light-chain variable region comprising an LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30-32 or with LCDR1, LCDR2 as shown in SEQ ID NOs: 30-32 LCDR variant with 3, 2 or 1 amino acid difference from LCDR3; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group consisting of the amino acid sequences of SEQ ID NOs: 33-35 or HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 33-35, respectively, 3, 2 Or an amino acid variant HCDR variant comprising a light-chain variable region comprising an LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 36-38 or LCDR1, LCDR2 as shown in SEQ ID NOs: 36-38 LCDR variant with 3, 2 or 1 amino acid difference from LCDR3; or
  • HCDR1, HCDR2 and HCDR3 selected from the amino acid sequences of SEQ ID NOs: 39-41 or HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 39-41, respectively, 3, 2 Or an amino acid variant HCDR variant comprising a light crystal chain variable region selected from the group consisting of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42-44 or with LCDR1, LCDR2 as shown in SEQ ID NO: 42-44 An LCDR variant with a difference of 3, 2 or 1 amino acid from LCDR3, respectively.
  • the CDRs of the monoclonal antibody or antigen-binding fragment including 3 heavy chain CDRs and 3 light chain CDRs) having 3, 2 or 1 amino acid differences are screened by affinity maturation methods CDR variants with 3, 2 or 1 amino acid differences were obtained.
  • the monoclonal antibody or antigen-binding fragment has an affinity (KD) to TIGIT of less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, or less than 10 -11 M.
  • the monoclonal antibody or antigen-binding fragment specifically binds to human TIGIT, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 15-17, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 18-20 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NOs: 21-23, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 24-26 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 27-29, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 30-32 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 33-35, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 36-38 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 39-41
  • the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 42-44 .
  • the monoclonal antibody is a recombinant antibody, preferably a recombinant antibody selected from the group consisting of a murine antibody, a chimeric antibody, and a humanized antibody.
  • sequences of the light and heavy chain FR regions on the humanized antibody light and heavy chain variable regions are derived from human germline light and heavy chains, or mutated sequences thereof, respectively.
  • the humanized antibody comprises a heavy chain variable region of SEQ ID NO: 45, 51, 56, 64 or 71 or a variant thereof; the variant is at SEQ ID NO
  • the heavy chain variable region sequence shown at 45, 51, 56, 64 or 71 has 1-10 amino acid changes.
  • the variant is a back mutation of 1-10 amino acids at the FR region of the heavy chain variable region set forth in SEQ ID NO: 45, 51, 56, 64 or 71;
  • the back mutation is selected from:
  • the humanized antibody comprises a heavy chain variable region selected from the group consisting of: (vi) SEQ ID NO: 45 or 50;
  • Ix a heavy chain variable region as set forth in any one of SEQ ID NOs: 64, 67, 68, 69 and 70;
  • the humanized antibody comprises a light chain variable region of SEQ ID NO: 46, 52, 57, 65 or 72 or a variant thereof; the back mutation is at SEQ ID NO
  • the light chain variable region shown at 46, 52, 57, 65 or 72 has a sequence of 1-10 amino acid changes.
  • the variant is a back mutation having 1-10 amino acids at the FR region position of the light chain variable region set forth in SEQ ID NO: 46, 52, 57, 65 or 72;
  • the back mutation is selected from:
  • humanized antibody comprises selected from the group consisting of:
  • Xiii a light chain variable region of any one of SEQ ID NOs: 57, 58, 59, and 60;
  • Xv a light chain variable region of any one of SEQ ID NOs: 72, 73 and 74.
  • humanized antibody comprises selected from the group consisting of:
  • Xix a heavy chain variable region of any one of SEQ ID NO: 64, 67, 68, 69 and 70 and a light chain variable region of SEQ ID NO: 65 or 66;
  • Xx a heavy chain variable region of any one of SEQ ID NO: 71, 75, 76 and 77 and a light chain variable region of any one of SEQ ID NOs: 72, 73 and 74.
  • a monoclonal antibody or antigen-binding fragment thereof wherein the antibody is a full length antibody, further comprising a human antibody constant region, wherein the heavy chain constant region is preferably a human IgG1, IgG2, IgG3, and IgG4 antibody heavy chain constant region More preferably, it comprises the human antibody heavy chain constant region of SEQ ID NO: 78 and the human light chain constant region of SEQ ID NO: 79.
  • a monoclonal antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region ( Diabodies), disulfide-stabilized V regions (dsFv) and antigen-binding fragments of peptides comprising CDRs.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region ( Diabodies), disulfide-stabilized V regions (dsFv) and antigen-binding fragments of peptides comprising CDRs.
  • a monoclonal antibody or antigen-binding fragment thereof which competes with the aforementioned monoclonal antibody or antigen-binding fragment thereof, in binding to human TIGIT.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the above, and one or more pharmaceutically acceptable carriers, diluents, buffers or agents Shape agent.
  • the amount of the monoclonal antibody or antigen-binding fragment thereof contained in the unit dose of the pharmaceutical composition is preferably from 0.1 to 2000 mg, more preferably from 0.1 to 1000 mg.
  • the present disclosure provides an isolated nucleic acid molecule encoding the above monoclonal antibody or antigen-binding fragment thereof.
  • the present disclosure provides a recombinant vector comprising the above nucleic acid molecule.
  • the present disclosure provides a host cell transformed with a recombinant vector according to the above, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
  • the present disclosure provides a method for producing the above monoclonal antibody or antigen-binding fragment thereof, the method comprising culturing the above host cell in culture to form and accumulate the above monoclonal antibody or antigen-binding fragment thereof, And recovering the monoclonal antibody or antigen-binding fragment thereof from the culture.
  • the present disclosure provides methods for detecting or determining human TIGIT, the method comprising the step of using the monoclonal antibody or antigen-binding fragment thereof described above.
  • the present disclosure provides an agent for detecting or determining human TIGIT, the reagent comprising the above monoclonal antibody or antigen-binding fragment thereof.
  • the present disclosure provides an agent for treating a disease associated with human TIGIT, comprising the above monoclonal antibody or antigen-binding fragment thereof, or comprising the above pharmaceutical composition, or comprising the nucleic acid molecule described above,
  • the disease is preferably a T cell dysfunction disorder, and T cell dysfunction is manifested in T cell depletion, which is to enhance the body's immune activity by enhancing NK cells and activating T cells, thereby achieving treatment or delay or alleviation of the disease, more preferably tumor, Cancer, immune disease or infectious disease.
  • cancer is preferably non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, Thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, adrenocortical carcinoma, liver hepatocellular carcinoma, pancreatic duct Adenocarcinoma, pheochromocytoma and ganglioneuroma, endometrial cancer and ovarian serous cystadenocarcinoma.
  • myeloma is preferably multiple myeloma (MM).
  • the immune disease is preferably arthritis, inflammatory bowel disease, psoriasis. Infectious diseases are preferably chronic viral infections.
  • the present disclosure provides a method of treating a disease associated with human TIGIT, the method comprising administering to a subject a pharmaceutically effective amount of the above monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, or Nucleic acid molecule for treating a human TIGIT-associated disease, preferably a T cell dysfunction, more preferably a tumor, cancer or infectious condition, most preferably a CD155 positive or PVR positive tumor, cancer, or immunity A disease or infectious condition.
  • the present disclosure provides the above monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, or the use of the above nucleic acid molecule for the preparation of a therapeutic agent for a disease associated with human TIGIT, preferably T A cell dysfunction condition, more preferably a tumor, cancer or infectious condition, most preferably a CD155 positive or PVR positive tumor, cancer, immune disease or infectious condition.
  • a therapeutic agent for a disease associated with human TIGIT preferably T A cell dysfunction condition, more preferably a tumor, cancer or infectious condition, most preferably a CD155 positive or PVR positive tumor, cancer, immune disease or infectious condition.
  • the present disclosure provides a method of treating a disease, the method comprising administering to a subject a pharmaceutically effective amount of the above monoclonal antibody or antigen-binding fragment thereof, or comprising the above pharmaceutical composition, or the isolated nucleic acid molecule
  • the disease is preferably a T cell dysfunction, more preferably a tumor, cancer or infectious condition, most preferably a CD155 positive or PVR positive tumor, cancer or infectious condition.
  • Figure 1 ELISA assay of TIGIT antibody binding to human TIGIT protein.
  • Figure 2 ELISA assay of TIGIT antibody binding to monkey TIGIT protein.
  • FIG. 3 Binding assay of TIGIT antibodies to CHO cells overexpressing human TIGIT.
  • Figure 4 Detection of binding affinity of TIGIT antibodies to human PBMC.
  • Figure 5A Detection of blocking effect of human TIGIT and CD155 binding by ch1708 and its humanized antibody
  • Figure 5B Detection of blocking effect of TIGIT humanized antibody on binding of human TIGIT to CD155.
  • FIG. 6 Blocking experiments of TIGIT antibodies binding to TIGIT antigens to CHO cells overexpressing CD155.
  • FIG. 7 Blocking experiments of TIGIT antibody binding of CD155 to CHO cells overexpressing TIGIT.
  • FIG. 8 Blocking experiments of TIGIT antibodies binding of TIGIT antigen to CHO cells overexpressing CD112.
  • FIG. 9 Binding endocytosis of TIGIT antibodies in TIGIT overexpressing CHO cells with endocytosis for 1 hour.
  • FIG. 10A TIGIT antibody promotes cell killing experiments of natural killer cells (NK);
  • Figure 10B TIGIT antibody promotes cell killing experiments of natural killer cells (NK).
  • Figure 11 TIGIT antibody versus PBMC-T lymphocyte activation assay.
  • Figure 12 Pharmacokinetic assay of TIGIT humanized antibodies in rats.
  • an “antibody” as used in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • murine antibody is in this disclosure a monoclonal antibody to human TIGIT prepared according to the knowledge and skill in the art.
  • the test subject is injected with the TIGIT antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine TIGIT antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprising a murine IgG1, IgG2 , heavy chain constant region of IgG3, IgG4 or variants thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • the human constant region gene is ligated into a chimeric gene, inserted into an expression vector, and finally expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the TIGIT chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the TIGIT chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or an amino acid mutation
  • An IgGl, IgG2 or IgG4 variant (such as a YTE mutation or a back mutation, S228P).
  • humanized antibody refers to the transplantation of murine CDR sequences into human antibody variable region frameworks, ie different types of human germline antibody framework sequences.
  • the antibody produced in it It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display.
  • the CDR sequence of the mouse in the TIGIT humanized antibody is selected from the group consisting of SEQ ID NOs: 15-44; the human antibody variable region framework is designed and selected, wherein the antibody heavy chain
  • the heavy chain FR region sequence on the variable region, the human germline heavy chain is selected from: (IGHV3-7*01 and hjh2), (IGHV1-46*01 and hjh4.1) and (IGHV1-69*02 and hjh4.
  • the combined sequence, and the human germline light chain are selected from: (IGKV1-39*02 and hjk2.1), (IGKV1-39*01 and hjk4.1) and (IGKV4-1*01 and hjk4.1) The combined sequence.
  • the human antibody variable region can be subjected to minimal reverse mutation (reversion mutation, that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues) to maintain activity.
  • the CDR graft can attenuate the affinity of the TIGIT antibody or antigen-binding fragment thereof to the antigen due to the framework residues that are in contact with the antigen. Such interactions can be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues involved in antigen binding from a non-human TIGIT antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related.
  • the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity.
  • Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • amino acid difference in a variant means that the variant protein or polypeptide has an amino acid change or mutation, including the basis of the original protein or polypeptide, compared to the original protein or polypeptide. Insertion, deletion or substitution of one or several amino acids occurs.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, TIGIT). It has been shown that fragments of full length antibodies can be utilized to achieve antigen binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of an antibody examples include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') 2 fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) A combination of two or more separate CDRs, optionally joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using a recombinant method such that they are capable of producing a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chains referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), inclusion Peptides of CDRs, etc.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (cleaving an amino acid residue at position 224 of the H chain), wherein the N-terminal side of the H chain About half of the entire L chain is bound by a disulfide bond.
  • the Fab of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human TIGIT and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof. Furthermore, the Fab can be produced by inserting a DNA encoding a Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express a Fab.
  • F(ab')2 is an antibody obtained by digesting the lower portion of two disulfide bonds in the IgG hinge region with an enzyme pepsin, having an molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions linked at the hinge position. Fragment.
  • the F(ab')2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human TIGIT and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with pepsin. Further, the F(ab') 2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2.
  • the Fab' of the present disclosure can be produced by treating F(ab')2 of the present disclosure which specifically recognizes TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting a DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • scFv antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996). , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by obtaining a cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes human TIGIT and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof, and constructs a DNA encoding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express an scFv.
  • a diabody is an antibody fragment in which an scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity.
  • the two antigens may be the same or different.
  • the diabody of the present disclosure can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes the human TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs a cDNA encoding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector such that the amino acid sequence of the peptide linker is 8 residues or less in length, and then the expression vector is introduced into a prokaryote or eukaryote In order to express double antibodies.
  • dsFv is obtained by linking a polypeptide in which one of amino acid residues in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues.
  • the amino acid residue substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present disclosure can be produced by obtaining a cDNA encoding VH and VL of a monoclonal antibody which specifically recognizes a human TIGIT and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof, and constructs a DNA encoding dsFv
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express dsFv.
  • a peptide comprising a CDR is constructed by one or more regions of a CDR comprising a VH or VL. Peptides comprising a plurality of CDRs can be joined directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present disclosure can be produced by constructing the DNA encoding the CDRs of the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes the human TIGIT and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express the peptide.
  • the CDR-containing peptide can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
  • antibody framework refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a TIGIT molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 -8 M, such as less than about 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind to TIGIT with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10 -8 M, 10 -9 M, or 10-10 M or less, for example, using surface plasma.
  • the body resonance (SPR) technique was measured in a BIACORE instrument.
  • an antigen binding protein eg, an antibody or immunologically functional fragment thereof
  • a reference antigen binding protein eg, a ligand or reference antibody
  • a common antigen eg, a TIGIT antigen or Specific binding of its fragment
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al, 1983, Methods in Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, eg, Kirkland et al, 1986, J. Immunol.
  • solid Direct labeling assay solid phase direct label sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling with I-125 label RIA (see, eg, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, eg, Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82).
  • the assay involves the use of a purified antigen (either on a solid surface or on the cell surface) that binds to a reference antigen binding protein with an unlabeled detection antigen binding protein and label.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein to be tested.
  • the antigen binding protein to be tested is present in excess.
  • An antigen binding protein identified by a competitive assay comprises: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding that binds to a neighboring epitope that is sufficiently close to the binding epitope of the reference antigen binding protein.
  • a competing antigen binding protein when present in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70. -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to a DNA molecule and an RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).
  • a mouse can be immunized with human TIGIT or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human TIGIT. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present disclosure, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any clinically measured extent.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount” effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • embodiments of the present disclosure may be ineffective in ameliorating the symptoms of each target disease
  • any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the present disclosure includes an agent for treating a disease associated with TIGIT, which comprises the monoclonal antibody of the present disclosure or an antibody fragment thereof as an active ingredient.
  • TIGIT a disease associated with TIGIT
  • a molecularly induced therapeutic response using the present disclosure can be followed by binding to human TIGIT and then repressing or inhibiting a T cell dysfunction, preferably a malignant tumor.
  • a cancer or infectious condition preferably a clinically responsive tumor or type of cancer observed in a clinical trial of an immunotherapeutic drug targeted at an immunotherapeutic checkpoint, most preferably a CD155 positive tumor, cancer or infectious condition.
  • the present disclosure relates to a method for immunodetection or assay of TIGIT, a reagent for immunodetection or assay of TIGIT, a method for immunodetection or assay of cells expressing TIGIT, and a diagnostic reagent for diagnosing a disease associated with TIGIT, It comprises as an active ingredient a monoclonal antibody or antibody fragment of the present disclosure which specifically recognizes human TIGIT and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof.
  • the method for detecting or measuring the amount of TIGIT may be any known method.
  • it includes immunodetection or assay methods.
  • the immunodetection or assay method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody.
  • immunoassay or assay methods include radioactive substance labeling immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods Wait.
  • TIGIT-related diseases can be diagnosed by detecting or measuring cells expressing TIGIT with the monoclonal antibodies or antibody fragments of the present disclosure.
  • a known immunodetection method can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or the like is preferably used. Further, a fluorescent antibody staining method or the like using the FMAT8100HTS system (Applied Biosystem) can be used.
  • a living sample for detecting or measuring TIGIT is not particularly limited as long as it has a possibility of including cells expressing TIGIT, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, Tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody of the present disclosure or an antibody fragment thereof may further contain an agent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on a desired diagnostic method.
  • Agents for performing antigen-antibody reactions include buffers, salts, and the like.
  • the reagents for detection include reagents commonly used in immunoassays or assay methods, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, and the like.
  • the TIGIT monoclonal antibody or antigen-binding fragment provided by the embodiments of the present disclosure has high specificity for TIGIT and high affinity with TIGIT, wherein the immunogenicity of the humanized antibody is greatly reduced, and the mouse antibody is completely retained. Specificity, high affinity and excellent in vitro and in vivo activity.
  • the TIGIT monoclonal antibody or antigen-binding fragment provided by the embodiments of the present disclosure has good metabolic kinetics on rats, exhibits a long half-life, and high bioavailability.
  • the TIGIT humanized antibody molecule provided by the embodiments of the present disclosure has good long-term stability, no obvious abnormal chemical modification, no obvious aggregation at high concentration, and high purity and thermal stability.
  • the TIGIT monoclonal antibody or antigen-binding fragment provided by the embodiments of the present disclosure has a good effect of enhancing NK cell and T cell activity.
  • the human TIGIT protein (Uniprot No.: Q495A1) was used as a template for the TIGIT of the present disclosure, and the amino acid sequences of the antigen and the detection protein of the present disclosure were designed, and different tags were fused on the basis of the TIGIT protein, and cloned into the pHr vector, respectively. (self-produced) or pXC-17.4 vector (LONZA), transient expression in 293 cells or stable expression and purification of CHO cells, obtaining a protein encoding the present disclosure and a protein for detection.
  • the following TIGIT antigens are not specifically described as human TIGIT.
  • TIGIT-mFc Fusion protein of TIGIT extracellular domain and mouse IgG2aFc segment: TIGIT-mFc for immunization and detection
  • the cross-hatched portion is the signal peptide and the italicized portion is the mFc.
  • TIGIT-Fc Fusion protein of TIGIT extracellular domain and human IgG1 Fc segment: TIGIT-Fc, for detection
  • the cross-hatched portion is the signal peptide and the italicized portion is the Fc.
  • TIGIT Full-length TIGIT: used to construct TIGIT overexpressing cell lines for detection
  • Fusion protein of cynoTIGIT extracellular domain and mouse IgG2aFc segment cynoTIGIT-mFc, for detection
  • the cross-hatched portion is the signal peptide and the italicized portion is the mFc.
  • ProteinG was selected for affinity purification of mouse hybridoma supernatant, and the cultured hybridoma was centrifuged to obtain a supernatant, and 10-15% by volume of 1 M Tris-HCl (pH 8.0-8.5) was added according to the supernatant volume. Clear pH.
  • the ProteinG column is washed with 3-5 times column volume with 6M hydrochloric acid, and then washed with pure water for 3-5 times column volume; using a buffer solution such as 1 ⁇ PBS (pH 7.4) as an equilibration buffer to equilibrate the column 3-5 Column volume; cell supernatant using low flow rate loading, control flow rate to maintain retention time of about 1min or longer; wash the column with 3-5 times column volume using 1 ⁇ PBS (pH 7.4) until UV absorption falls back to Baseline; sample elution with 0.1 M acetic acid/sodium acetate (pH 3.0) buffer, and elution peaks were collected according to UV detection.
  • a buffer solution such as 1 ⁇ PBS (pH 7.4) as an equilibration buffer to equilibrate the column 3-5 Column volume; cell supernatant using low flow rate loading, control flow rate to maintain retention time of about 1min or longer; wash the column with 3-5 times column volume using 1 ⁇ PBS (pH 7.
  • solution displacement can be carried out by methods well known to those skilled in the art, such as ultrafiltration filtration using ultrafiltration tubes and solution displacement to a desired buffer system, or replacement with a molecular exclusion such as G-25 desalting.
  • the cell culture supernatant expressing the Fc fusion protein or antibody is first subjected to high-speed centrifugation to collect the supernatant.
  • the ProteinA affinity column was washed with 3-5 column volumes using 6 M hydrochloric acid, and then washed 3-5 times column volume with pure water.
  • the column was equilibrated with 3-5 column volumes using a buffer solution such as 1 x PBS (pH 7.4) as an equilibration buffer.
  • the cell supernatant was loaded with a low flow rate, and the flow rate was controlled so that the retention time was about 1 min or longer.
  • the column was washed 3-5 times with 1 ⁇ PBS (pH 7.4) until the UV absorption dropped back to the baseline. .
  • the sample was eluted with 0.1 M acetic acid/sodium acetate (pH 3.0-3.5) buffer, and the elution peak was collected according to the ultraviolet detection.
  • the eluted product was quickly adjusted to pH 5-6 by using 1 M Tris-HCl (pH 8.0). Save.
  • solution displacement can be carried out by methods well known to those skilled in the art, such as ultrafiltration filtration using ultrafiltration tubes and solution displacement to a desired buffer system, or replacement with a molecular exclusion such as G-25 desalting.
  • Anti-human TIGIT monoclonal antibodies are produced by immunizing mice.
  • Experimental SJL white mice female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized according to the following protocol.
  • the immunizing antigen is the human TIGIT extracellular domain with mFc (SEQ ID NO: 1).
  • Immunization program use Gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) adjuvant cross-immunization.
  • Antigen and adjuvant ( Gold Adjuvant) ratio of 1:1, antigen and adjuvant (Thermo Alum) ratio is 3:1, 50 ⁇ g/only/time (first immunization), 25 ⁇ g/only/time (enhanced immunization).
  • the antigen was emulsified and inoculated for 0, 14, 28, 42, and 56 days. On day 0, 50 ⁇ g/cell of emulsified antigen was injected intraperitoneally (IP).
  • IP intraperitoneally
  • Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in complete medium (DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 ⁇ 6/ml, and 100 ⁇ l/well in 96 wells. After incubating for 3-4 days at 37 ° C, 5% CO 2 in the plate, HAT complete medium was supplemented with 100 ⁇ l/well, and culture was continued for 3-4 days until a needle-like clone was formed.
  • complete medium DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI was added, and cultured at 37 ° C, 5% CO 2 for 3 days, and then subjected to ELISA.
  • Hybridoma culture supernatants were detected by binding ELISA according to the growth density of hybridoma cells.
  • the positive well cell supernatants detected by ELISA were combined for cell binding assay and cell blocking assay.
  • the well cells that were positive for both binding and blocking were expanded in a timely manner for cryopreservation and two to three subclones until a single cell clone was obtained.
  • the subcloned cells are also subjected to TIGIT binding ELISA, HTRF blocking assay, cell binding assay and cell blocking assay.
  • the hybridoma clones were screened by the above experiments, and the antibody was further prepared by serum-free cell culture, and the antibody was purified according to the purification example for use in the test examples.
  • the sequence of cloning from a positive hybridoma is as follows. Hybridoma cells in logarithmic growth phase were collected, using Trizol (Invitrogen, Cat No.15596-018) according to kit instructions an RNA extraction step, with PrimeScript TM Reverse Transcriptase reverse transcription kit (Takara, Cat No.2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced. The amino acid sequence of the variable region of the antibody corresponding to the positive clone m17 obtained from the obtained DNA sequence was as follows:
  • the heavy chain and light chain variable region germline genes with high homology to the murine antibody were selected as templates, and the murine antibody was
  • the CDRs are each grafted into the corresponding human template to form a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the backbone sequence are back-mutated to the amino acids corresponding to the murine antibody to ensure the original affinity, that is, the humanized anti-TIGIT monoclonal antibody is obtained.
  • the determination of the amino acid residues in the CDR regions is determined and annotated by the Kabat numbering system.
  • the light heavy chain variable region of the murine antibody is ligated to the light heavy chain constant region of the human antibody to form a chimeric antibody, and the chimeric antibody corresponding to the m1707 antibody is named ch1707, and other antibodies are analogized.
  • the humanized light chain template of the murine antibody m1707 is IGKV1-39*02 and hjk2.1, and the humanized heavy chain template is IGHV3-7*01 and hjh2. After humanization, the humanized antibody h1707 is obtained.
  • the sequence of the sourced variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • This table represents the sequence obtained by various combinations of mutations. As indicated by h1707-04, there are four back mutations of the light chain L2 and the heavy chain H2 on the humanized antibody h1707-04. Other analogies.
  • the humanized light chain template of the murine antibody m1708 is IGKV1-39*01 and hjk4.1, and the humanized heavy chain template is IGHV1-46*01 and hjh4.1. After humanization, the humanized antibody h1708 is obtained.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • A43S indicates that the 43-position A is mutated back to S according to the natural sequence of the amino acid sequence. Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • This table represents the sequence obtained by various combinations of mutations. As indicated by h1708-05, there are two back mutations of the light chain L2 and the heavy chain H2 on the humanized antibody h1708-05. Other analogies.
  • the humanized light chain template of the murine antibody m1709 was IGKV1-39*01 and hjk4.1, and the humanized heavy chain template was IGHV1-46*01 and hjh4.1. After humanization, the humanized antibody h1709 was obtained.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • This table represents the sequence obtained by various combinations of mutations. As indicated by h1709-06, there are three back mutations of the light chain L2 and the heavy chain H2 on the humanized antibody h1709-06. Other analogies.
  • the humanized light chain template of the murine antibody m1710 was IGKV1-39*01 and hjk4.1, and the humanized heavy chain template was IGHV1-46*01 and hjh4.1. After humanization, the humanized antibody h1710 was obtained.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • A43S indicates that the 43-position A is mutated back to S according to the natural sequence of the amino acid sequence. Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • This table represents the sequence obtained by various combinations of mutations. As indicated by h1710-07, there are four back mutations of the light chain L2 and the heavy chain H2 on the humanized antibody h1710-07. Other analogies.
  • the humanized light chain template of the murine antibody m1711 was IGKV4-1*01 and hjk4.1, and the humanized heavy chain template was IGHV1-69*02 and hjh4.1. After humanization, the humanized antibody h1711 was obtained.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • This table represents the sequence obtained by various combinations of mutations. As indicated by h1711-06, a back-mutated antibody composed of a light chain L2 and a heavy chain H2 on the humanized antibody h1711-06. Other analogies.
  • Each of the above heavy chain variable regions can be recombinantly expressed with the heavy chain constant region sequence (IgG4, with the S228P mutation) as set forth in SEQ ID NO: 78 to give the final entire heavy chain sequence.
  • Each of the above light chain variable regions is recombinantly expressed with the light chain constant region sequence (kappa chain) as set forth in SEQ ID NO: 79 to give the final intact heavy light chain sequence.
  • the heavy and light chain variable regions described above can also be recombined with other heavy chain constant regions or mutated IgG family constant regions of the IgG family well known in the art to form intact antibody heavy light chain sequences.
  • An exemplary constant region sequence is as follows:
  • IgG4 heavy chain constant region with S228P mutation is S228P mutation:
  • the positive control antibody is 22G2-H3Q
  • the VH and VL sequences are from US20160176963A1 (SEQ ID NOS: 8 and 9 of US20160176963A1, respectively), and the heavy chain constant region and light of SEQ ID NO: 78 and SEQ ID NO: 79, respectively.
  • the constant region of the chain forms an intact full length antibody.
  • the specific sequences of VH and VL of 22G2-H3Q are as follows:
  • Test Example 1 ELISA assay of TIGIT antibody binding to human TIGIT protein
  • the binding capacity of the anti-TIGIT antibody was detected by an ELISA assay of the antibody with the human TIGIT protein.
  • the TIGIT fusion protein with Fc or mFc tag is immobilized into a 96-well microtiter plate by binding to an anti-Fc or mFc antibody coated in an ELISA plate, and the signal intensity after antibody addition is used to judge the antibody and TIGIT.
  • the positive control molecule is 10A7 hIgG4 (wherein the light heavy chain variable region sequence of 10A7 IgG4 is derived from SEQ ID NOS: 21 and 22 of US20130251720A1, and the light and heavy chain variable regions are ligated to form the full-length antibody in the light and heavy chain constant regions, respectively
  • the negative control is a human IgG4 antibody (hIgG4) that does not bind to TIGIT, and the specific experimental method is as follows:
  • Goat anti-human Fc antibody Jackson Immuno Research, Cat No. 109-005-008
  • goat anti-mouse Fc antibody Sigma, Cat No.
  • PBS Sthai Yuanpei, Cat No. B320
  • M3534-1ML was diluted to a concentration of 2 ⁇ g/ml, and added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) in a volume of 50 ⁇ l/well, and placed in an incubator at 37 ° C for 2 hours. After discarding the liquid, add 200 ⁇ l/well of 5% skim milk (BD skim milk, Cat No.
  • Test Example 2 ELISA assay of TIGIT antibody binding to cynomolgus monkey TIGIT protein
  • Monkey cross-binding of anti-TIGIT antibodies was detected by ELISA assay of antibodies with cynomolgus TIGIT protein.
  • the cynomolgus TIGIT fusion protein with Fc or mFc tag is immobilized into a 96-well microtiter plate by binding to an anti-Fc or mFc antibody coated in a ELISA plate, and the signal strength after antibody addition is used for judgment.
  • the binding activity of the antibody to the cynomolgus monkey TIGIT is as follows:
  • Goat anti-human Fc antibody Jackson Immuno Research, Cat No. 109-005-008
  • goat anti-mouse Fc antibody Sigma, Cat No.
  • PBS Sthai Yuanpei, Cat No. B320
  • M3534-1ML was diluted to a concentration of 2 ⁇ g/ml, and added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) in a volume of 50 ⁇ l/well, and placed in an incubator at 37 ° C for 2 hours. After discarding the liquid, add 200 ⁇ l/well of 5% skim milk (BD skim milk, Cat No.
  • the cynomolgus monkey TIGIT-Fc fusion protein (internal production) or the cynomolgus TIGIT-mFc fusion protein (internal production) to 0.5 ⁇ g/ml was incubated at 37 ° C for 1 hour or at 4 ° C overnight. After the incubation, discard the reaction solution in the plate, wash the plate with PBST for 5 times, and add 50 ⁇ l/well of the antibody to be tested (hybridoma purified antibody or humanized antibody) diluted with the sample dilution. Incubate for 1 hour at 37 ° C in the incubator.
  • the absorbance was read at a wavelength of 450 nm by a microplate reader (Thermo scientific Multiskan MK3), and the binding EC50 value of the TIGIT antibody to the monkey TIGIT was calculated by analyzing the data with GraphPad Prism 5. The results are shown in Figure 2.
  • Test Example 3 Binding experiment of TIGIT antibody and human TIGIT overexpressing CHO cells
  • the binding capacity of the anti-TIGIT antibody was detected by binding experiments of the antibody to CHO cells overexpressing the TIGIT protein.
  • the TIGIT full-length plasmid was transformed into CHO cells by electroporation for two weeks, and the expression level of TIGIT was examined. After the overexpressing cells were fixed in the 96-well plate bottom, the signal intensity after the antibody was added was used to judge the binding activity of the antibody and TIGIT overexpressing CHO cells.
  • the specific experimental methods are as follows:
  • the cells were seeded at a density of 5 x 10 ⁇ 5/ml, 100 ⁇ l/well in 96-well plates overnight. The supernatant was discarded, washed three times with PBS, and then 100 ⁇ l/well of cell immunofixation solution (Beyotime, Cat No. P0098) was added and fixed at room temperature for half an hour, and washed with PBS four times. After discarding the liquid, 200 ⁇ l/well of a 5% skim milk (BD skim milk, Cat No. 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 3 hours for blocking.
  • a 5% skim milk BD skim milk, Cat No. 232100
  • the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), and 50 ⁇ l/well of the antibody to be tested diluted with the sample diluent was added (hybridoma purification).
  • PBST buffer pH 7.4 PBS containing 0.05% tweeen-20
  • Antibody or humanized antibody incubated in a 37 ° C incubator for 1 hour. After the incubation, the plate was washed 5 times with PBST, and 50 ⁇ l/well of HRP-labeled goat anti-mouse secondary antibody diluted with sample dilution (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson) was added.
  • Immuno Research Cat No.
  • Test Example 4 Binding experiment of TIGIT antibody and human PBMC
  • the binding of anti-TIGIT antibodies was detected by binding experiments of antibodies to human activated PBMCs in vitro.
  • Human PBMC was activated by the method of superantigen S. aureus enterotoxin B (SEB) stimulation, and the intensity of the fluorescent signal after antibody addition was used to judge the binding activity of the antibody and activated human PBMC.
  • SEB superantigen S. aureus enterotoxin B
  • PBMC fresh blood was obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies) to obtain PBMC, which was cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS and added with superantigen S. aureus enterotoxin B. (SEB) 500 ng/ml, cultured for 4 days at 37 ° C, 5% CO 2 .
  • SEB superantigen S. aureus enterotoxin B.
  • the activated PBMC cells were seeded at a density of 5 ⁇ 10 ⁇ 6 / ml, 100 ⁇ l / well in a 96-well round bottom plate (Corning, Cat No. 32915001), centrifuged at 1500 rpm for 5 minutes in a centrifuge (Beckman Coulter, Allegra X-15R Centrifuge). Discard the supernatant; resuspend the cells in 200 ⁇ l PBS, centrifuge, discard the supernatant, and repeat.
  • the cells were resuspended by adding 100 ⁇ l/well of the antibody solution to be tested which was diluted with a sample dilution (pH 7.4 PBS containing 1% BSA), and incubated at 4 ° C for 1 hour. After the incubation, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, wash the cells twice with the sample dilution, and resuspend the cells by adding 100 ⁇ l of PE-goat anti-human IgG (Jackson ImmunoResearch, 109-115-098) dilution, incubate at 4 °C. 1 hour.
  • PE-goat anti-human IgG Jackson ImmunoResearch, 109-115-098
  • the affinity of the humanized anti-TIGIT antibody to be tested with human and monkey TIGIT was determined using a Biacore, GE instrument.
  • Human anti-capture antibody was covalently coupled using the Protein A biosensor chip (Cat. #29127556, GE) for affinity capture or according to the method in the Human Anti-Capture Kit (Cat. #28-9538-28, GE) instructions.
  • the affinity capture On the biosensor chip (Cat.#28-9538-28, GE), the affinity captures a certain amount of the antibody to be tested, and then flows through the surface of the chip through a series of concentration gradients of human, monkey TIGIT antigen, human source.
  • TIGIT can be selected from Yishen Shenzhou Biotechnology Co., Ltd. (Cat.10917-H08-H, Sino.Biol), and the monkey TIGIT is purified from the expression of Example 1 and Example 2.
  • the reaction is detected in real time using Biacore instrument (Biacore T200, GE).
  • Biacore T200, GE Biacore T200, GE
  • the signal thus obtains a binding and dissociation curve.
  • the biochip was washed and regenerated using a regeneration solution disposed in a human anti-capture kit or a glycine-hydrochloric acid regeneration solution (Cat. #BR-1003-54, GE) at pH 1.5.
  • the amino coupling kit used in the experiment was purchased from GE (Cat. #BR-1000-50, GE) and the buffer was HBS-EP+10 ⁇ buffer solution (Cat.#BR-1006-69, GE). Dilute to 1 x (pH 7.4) with DIWater.
  • Test Example 6 Blocking test of TIGIT antigen binding to TIGIT antigen to CD155 protein
  • the blocking ability of the anti-TIGIT antibody was detected by an HTRF assay in which the antibody blocks TIGIT binding to CD155 protein.
  • the pair of donors and receptors with TIGIT-Fc and biotinylated CD155 were used with Pab Anti-Human IgG-Tb (Cisbio, Cat No. 61HFCTAA) and Streptavidin-XL665 (Cisbio, Cat No. 610 SAXLA). CD-050/CF) binding, or Pab Anti-mouse-IgG-XL665 (Cisbio, Cat No. 61PAMXLA) and Streptavidin-Tb (Cisbio, Cat No.
  • test antibody Hybridoma purified antibody or humanized antibody
  • dilution pH 7.4 PBS containing 1% BSA
  • 384-well experimental plate Cornning, Cat No. 3706
  • Test Example 7 Blocking experiment of TIGIT antigen binding to TIGIT antigen and CHO cells overexpressing CD155
  • the blocking ability of the anti-TIGIT antibody was detected by an ELISA assay in which the antibody blocked TIGIT binding to CHO cells overexpressing CD155.
  • the CD155 full-length plasmid was transfected into CHO cells by electroporation for two weeks under pressure screening, and the expression level of CD155 was examined. After the overexpressing cells were fixed in the bottom of the 96-well plate, TIGIT was pre-incubated with the diluted anti-TIGIT antibody and added to the well plate. After the secondary antibody was added, the signal intensity was used to judge the antibody blocking TIGIT and over-expressing CD155.
  • the ability of CHO cells to bind, the specific experimental methods are as follows:
  • CD155-CHO cells were seeded at a density of 5 x 10 ⁇ 5/ml, 100 ⁇ l/well in 96-well plates overnight. The supernatant was discarded, washed three times with PBS, and then fixed in a 100 ⁇ l/well cell immunofixation solution (Beyotime, Cat No. P0098) at room temperature for half an hour, and washed four times with PBS. After discarding the liquid, 200 ⁇ l/well of a 5% skim milk (BD skim milk, Cat No. 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 3 hours for blocking.
  • a 5% skim milk BD skim milk, Cat No. 232100
  • the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tweeen-20), and then 50 ⁇ l/well diluted with the sample diluent (pH 7.4 PBS containing 1% BSA) was added.
  • PBST buffer pH 7.4 PBS containing 0.05% tweeen-20
  • 50 ⁇ l/well diluted with the sample diluent pH 7.4 PBS containing 1% BSA
  • the absorbance was read at a wavelength of 450 nm by Thermo Scientific Multiskan (MK3), and the data was analyzed by GraphPad Prism 5 to calculate the blocking effect of the TIGIT antibody on the binding of the antigen to CHO cells overexpressing CD155. The results are shown in Figure 6.
  • Test Example 8 Blocking experiment of binding of CD155 protein to CHO cells overexpressing TIGIT by TIGIT antibody
  • the blocking ability of the anti-TIGIT antibody was detected by FACS experiments in which the antibody blocked the binding of CD155 to TIGIT overexpressing CHO cells.
  • the TIGIT full-length plasmid was transfected into CHO cells by electroporation for two weeks under pressure screening, and the expression level of TIGIT was examined.
  • the pre-expressed cells were pre-incubated with different concentrations of anti-TIGIT antibodies, and then fluorescently labeled with CD155-Fc was added for incubation. The strength of the signal was used to judge the ability of the antibody to block the binding of CD155 to CHO cells overexpressing TIGIT.
  • the experimental method is as follows:
  • CF TM 633 (Sigma Aldrich, Cat No.MX633S100) labeled CF TM 633 (Sigma Aldrich, Cat No.MX633S100) a fluorescent dye. After incubation CF TM 633 was then added fluorescent dyes, in the dark room temperature for 30 min; the CD155-Fc dissolved in PBS to a concentration of 0.5-1mg / ml, was added 9 times the sample volume of 10 ⁇ Mix-n-Stain Reaction Buffer, mix The fluorescent label is completed.
  • TIGIT-CHO cells were seeded at a density of 5 ⁇ 10 ⁇ 6 / ml, 100 ⁇ l / well in a 96-well round bottom plate (Corning, Cat No. 32915001), centrifuged at 1500 rpm for 5 minutes in a centrifuge (Beckman Coulter, Allegra X-15R Centrifuge). Discard the supernatant; resuspend the cells in 200 ⁇ l PBS, centrifuge, discard the supernatant, and repeat.
  • the cells were resuspended by adding 100 ⁇ l/well of the antibody solution to be tested which was diluted with a sample dilution (pH 7.4 PBS containing 1% BSA), and incubated at 4 ° C for 1 hour. After the incubation, the cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with the sample dilution, and then resuspended by adding 100 ⁇ l of 2 ⁇ g/ml CF TM 633 fluorescently labeled CD155-Fc solution, and incubated at 4 ° C for 1 hour.
  • a sample dilution pH 7.4 PBS containing 1% BSA
  • h1707-02, h1708-04, h1709-10, h1710-01, h1711-04 antibodies were able to block the binding of CD155 to TIGIT overexpressing CHO cells.
  • Test Example 9 Blocking experiment of TIGIT antigen binding to TIGIT antigen and CHO cells overexpressing CD112
  • the blocking ability of the anti-TIGIT antibody was detected by FACS experiments in which the antibody blocked the binding of TIGIT to CD112 overexpressing CHO cells.
  • the CD112 full-length plasmid was transfected into CHO cells by electroporation for two weeks, and the expression level of CD112 was measured.
  • the TIGIT-mFc protein was pre-incubated with dilute different concentrations of anti-TIGIT antibody and added to CD112-overexpressing CHO cells for incubation.
  • the antibody labeled with PE label was used to detect the intensity of TIGIT signal and was used to determine the antibody blocking TIGIT and overexpression.
  • the ability of CD112 to bind CHO cells is as follows:
  • Test Example 10 Binding endocytosis assay of TIGIT antibody over TIGIT overexpressing CHO cells
  • TIGIT antibodies To investigate the ability of TIGIT antibodies to endocytosis upon binding to cell surface antigens, FACS experiments were performed on CHO cells expressing full length TIGIT for endocytosis of TIGIT antibodies.
  • the specific experimental methods are as follows:
  • TIGIT-CHO cells were seeded at a density of 2 ⁇ 10 ⁇ 6/ml, 100 ⁇ l/well in a 96-well round bottom plate (Corning, Cat No. 32915001), and centrifuged at 1500 rpm for 5 minutes in a centrifuge (Beckman Coulter, Allegra X-15R Centrifuge). The supernatant was discarded; the cells were resuspended in 200 ⁇ l of 1% BSA, centrifuged, and the supernatant was discarded and repeated.
  • the humanized antibody sample was diluted to a concentration of 4 ⁇ g/mL with 1% BSA, and the cells were resuspended in 100 ⁇ l/well and incubated on ice for 1 hour.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • WiDr lactate dehydrogenase
  • the human colorectal cancer cell line WiDr was cultured in MEM medium, and 10% (v/v) fetal bovine serum (FBS) was added to the medium, and cultured at 37 ° C under 5% CO 2 .
  • Fresh blood was obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies) to obtain PBMC, and human primary NK cells were extracted from freshly isolated PBMC (Miltenyi, CAT#130-092-657) and cultured in RPMI 1640 medium. 10% (v/v) FBS was added to the medium, and cultured at 37 ° C under 5% CO 2 .
  • Human primary NK cells were seeded into a 6-well cell culture plate at a cell density of approximately 2 ⁇ 10 ⁇ 6/ml. After addition to 100 U/mL human IL-2 overnight, the cells were washed with phenol-free red RPMI 1640 medium and resuspended. The cells were seeded in a 96-well U-bottom plate at a cell density of approximately 3 x 10 ⁇ 5/well, and a gradient-diluted antibody sample (diluted with PBS) or an equivalent amount of homo-IgG was added as a blank control.
  • the target cell WiDr was co-cultured with human primary NK cells at a ratio of 1:1, and cultured at 37 ° C, 5% CO 2 incubator for 4 h, and the cell culture supernatant was collected.
  • CytoTox The Non-Radioactive Cytotoxicity Assay (Promega, CAT #G1780) specification method detects the level of LDH secretion in the cell culture supernatant.
  • TIGIT humanized candidate antibodies h1707-02, h1708-04, h1710-01 and other antibodies can enhance the killing of target cells by human primary NK cells to varying degrees, and Drug concentration dose effect.
  • Test Example 12 PBMC-T lymphocyte activation assay
  • PBMC peripheral blood mononuclear cells
  • Fresh blood PBMC were obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies), and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, and cultured at 37 ° C under 5% CO 2 .
  • the freshly purified PBMC was adjusted to a density of 2 ⁇ 10 6 /ml in RPMI 1640 medium, and 25 ⁇ l of tuberculin was added to 20 mL of the cell suspension, and cultured in a 37 ° C, 5% CO 2 incubator for 5 days.
  • CD155 recombinant CD155/PVR Protein, R&D, 2530-CD-050/CF
  • R&D Recombinant CD155/PVR Protein, R&D, 2530-CD-050/CF
  • the cultured cells were collected and centrifuged, washed once with PBS, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1 ⁇ 10 6 /ml, and seeded into a CD155-coated 96-well cell culture plate. 90 ⁇ l per well.
  • a gradient diluted antibody sample (diluted with PBS) or an equivalent amount of isotype IgG was added as a blank control, 10 ⁇ l per well.
  • the cell culture plates were incubated at 37 ° C for 3 days in a 5% CO 2 incubator.
  • TIGIT humanized candidate antibodies h1708-04, h1710-01 and other antibodies can enhance the secretion of cytokine IFN- ⁇ by activated primary T lymphocytes to varying degrees, and there is a drug concentration dose effect.
  • SD male rats weighing 180-240 g, were purchased from Xipuer-Beikai Experimental Animal Co., Ltd. Free feed and water during feeding, laboratory environment adaptive feeding no less than 3 days, 12/12 hour light/dark cycle adjustment, temperature 16-26 ° C, relative humidity 40-70%.
  • One day before the start of the experiment SD rats were numbered and randomly divided into groups of 3 each.
  • the rats in the five groups were injected intravenously or subcutaneously with the test drugs h1707-02, h1708-04, h1710-01, h1711-04 and h1709-10, 22G2-H3Q as positive controls at a dose of 3 mg/kg.
  • the injection volume was 5 ml/kg.
  • Intravenous administration was performed at various time points before and 5 minutes, 8h, 1d, 2d, 4d, 7d, 10d, 14d, 21d, and 28d.
  • the blood was collected by subcutaneous injection at the time points before and after 1 h, 4 h, 8 h, 1 d, 2 d, 4 d, 8 d, 11 d, 14 d, 21 d, and 28 d.
  • Each animal took 0.2 ml of whole blood without anticoagulant. After taking blood, it was placed at 4 ° C for 30 min, centrifuged at 1000 g for 15 min, and the supernatant was placed in an EP tube and stored at -80 ° C.
  • the antibody concentration in the serum was measured by an ELISA method (see Test Example 1 of Example 3), and the pharmacokinetic parameters of the test drug were calculated using Winnolin software. The main pharmacokinetic results of the obtained part are shown in Figure 12.
  • Test Example 15 Monitoring the purity of the sample by SEC-HPLC to investigate the cycle stability under a certain concentration condition
  • Exemplary conditions such as controlling the sample concentration at about 50 mg/ml, comparing different antibodies at -80 ° C in 10 mM PBS (pH 7.4), 15 mM His (pH 6.0), 10 mM Acetate acid (pH 5.2) and the like. Repeated freezing and thawing 5 times and stability at 4 ° C and 40 ° C for 28 days. The purity of the antibody was detected by Xbridge protein BEH SEC 200A (Waters) HPLC column. H1707-02, h1708-04, h1709-10, h1710-01, h1711-04, etc. showed good stability, and accelerated SEC purity at 40 °C per month. No significant changes.
  • 500 ⁇ g of the antibody to be tested was dissolved in 500 ⁇ l of PBS pH 7.4 at 40 ° C in water bath; samples were taken at 0, 14, and 28 days, respectively, for enzymatic hydrolysis experiments. 100 ⁇ g samples taken at different time points were dissolved in 100 ⁇ l of 0.2 M His-HCl, 8 M Gua-HCl, pH 6.0 solution, 3 ⁇ l of 0.1 g/mL DTT, 50 ° C water bath for 1 hour, and then 0.02 M His-HCl, pH. The solution of 6.0 was ultrafiltered twice, and 3 ⁇ L of 0.25 mg/mL trypsin was added, and the mixture was hydrolyzed overnight at 37 ° C in a water bath.
  • the Agilent 6530 Q-TOF was analyzed by LC-MS. The results showed that h1707-02, h1708-04, h1709-10, h1710-01, and h1711-04 did not show significant water loss and oxidation after being accelerated for 40 months. Unstable changes such as deamidation suggest good chemical stability of the molecule.

Abstract

一种TIGIT 抗体、其抗原结合片段及医药用途。进一步地，还涉及包含所述TIGIT 抗体CDR 区的鼠源抗体、嵌合抗体、人源化抗体，以及包含TIGIT 抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。特别地，还涉及一种人源化的TIGIT 抗体在制备用于治疗TIGIT 相关的疾病或病症的药物中的用途。

Description

TIGIT抗体、其抗原结合片段及医药用途
本申请是基于，并且要求以下文件的优先权，中国申请号为201710908565.3，申请日为2017年9月29日，该申请的公开内容都通过引用并入到了本申请当中。
技术领域
本公开涉及TIGIT抗体以及其抗原结合片段，进一步地，本公开还涉及包含所述TIGIT抗体CDR区的嵌合抗体、人源化抗体，本公开还涉及包含所述TIGIT抗体及其抗原结合片段的药物组合物，以及其作为TIGIT相关疾病诊断剂和治疗药物的用途。
背景技术
近年来，针对于免疫细胞共抑制受体的免疫卡控点治疗在肿瘤免疫治疗中取得了巨大进展，发现和验证新的共抑制性受体成为一个全球竞争性的热点。T细胞是免疫反应的关键介导者，T细胞的活化依赖TCR信号和共刺激信号。共刺激信号则是T细胞活化的限制信号，其功能失调参与自身免疫疾病的发生(Immunol Rev,2012,248:122-139；Autoimmun Rev,2013,12:1171-1176)。TIGIT(T cell immunoglobulin and ITIM domain)是新发现的位于NK细胞和T细胞表面的共抑制信号分子，与T细胞、NK细胞及树突状细胞DC等的功能调节密切相关。
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。TIGIT分子属于免疫球蛋白超家族IgSF的一员，其结构较为保守，多种哺乳动物中都发现了其同源分子，人类TIGIT分子与猴、狗和小鼠的TIGIT分子分别具有88％、67％和58％的同源性(Nat Immunol，2009，10(1):48－57)。
TIGIT分子主要表达于T细胞和NK细胞表面(Nat Immunol,2009,10:48-57)。
Figure PCTCN2018108246-appb-000001
T细胞和静息记忆T细胞均低表达TIGIT，体外活化后表达上调(J Immunol,2012,188:3869-3875)。NK细胞表面TIGIT则有较高水平的表达(Proc Natl Acad Sci USA，2009，106(42):17858－17863)。TIGIT是极具潜力的免疫治疗新靶点。已有研究表明特异性地阻断TIGIT的单克隆抗体，在动物模型中显示了显著的抗肿瘤效果(Martinet and Smyth 2015)。TIGIT抗体和PD-1抗体联用，能够促进CD8T细胞对HIV和黑色素瘤的杀伤功能，这一效应随着对CD226的阻断而消失(Chew,Fujita et al.2016)。目前国内外还没有阻断TIGIT的单克隆抗体药物上市，因此亟须开展高效特异性TIGIT单克隆抗体的研发。
目前，已有WO2009126688、WO2014089113、WO2015009856、WO2015143343、WO2015174439、WO2017053748、WO2017030823、WO2016106302、US20160176963、US20130251720等专利报道了TIGIT的抗体及相关应用。但目前为止，尚无TIGIT抗体应用于临床，仍需开发新的，更适于临床应用的TIGIT抗体。
发明内容
本公开提供与TIGIT的胞外区的氨基酸序列或三维结构特异性结合的单克隆抗体或抗原结合片段(也可称TIGIT结合分子)。
一方面，提供一种单克隆抗体或其抗原结合片段，所述单克隆抗体或抗原结合片段特异性结合人TIGIT，所述单克隆抗体包含重链可变区和轻链可变区，其中：
(i)重链可变区包含选自如SEQ ID NO：15、16和17氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：15、16和17所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：18、19和20氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：18、19和20所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
(ii)重链可变区包含选自如SEQ ID NO：21-23氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：21-23所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：24-26氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：24-26所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
(iii)重链可变区包含选自如SEQ ID NO：27-29氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：27-29所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：30-32氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：30-32所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
(iv)重链可变区包含选自如SEQ ID NO：33-35氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：33-35所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：36-38氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：36-38所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
(v)重链可变区包含选自如SEQ ID NO：39-41氨基酸序列所示的HCDR1、 HCDR2和HCDR3或与SEQ ID NO：39-41所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：42-44氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：42-44所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体。
在一些实施方式中，所述单克隆抗体或抗原结合片段的CDR(包括3个重链CDR和3个轻链CDR)具有3，2或1个氨基酸差异的CDR变体是经亲和力成熟方法筛选获得的具有3，2或1个氨基酸差异的CDR变体。
在一些实施方式中，所述单克隆抗体或抗原结合片段与TIGIT的亲和力(KD)小于10 -8M、小于10 -9M、小于10 -10M或小于10 -11M。
在一些实施方式中，所述单克隆抗体或抗原结合片段特异性结合人TIGIT，所述单克隆抗体包含重链可变区和轻链可变区，其中：
(vi)重链可变区包含如SEQ ID NO：15-17所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：18-20所示的LCDR1、LCDR2和LCDR3区；或
(vii)重链可变区包含如SEQ ID NO：21-23所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：24-26所示的LCDR1、LCDR2和LCDR3区；或
(viii)重链可变区包含如SEQ ID NO：27-29所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：30-32所示的LCDR1、LCDR2和LCDR3区；或
(ix)重链可变区包含如SEQ ID NO：33-35所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：36-38所示的LCDR1、LCDR2和LCDR3区；或
(x)重链可变区包含如SEQ ID NO：39-41所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：42-44所示的LCDR1、LCDR2和LCDR3区。
在一些实施方式中，其中所述单克隆抗体是重组抗体，优选选自鼠源抗体、嵌合抗体、人源化抗体的重组抗体。
在一些实施方式中，其中所述的人源化抗体轻链和重链可变区上的轻链和重链FR区序列分别来源于人种系轻链和重链或其突变序列。
在一些实施方式中，其中所述的人源化抗体含有SEQ ID NO:45、51、56、64或71所示的重链可变区或其变体；所述变体是在SEQ ID NO:45、51、56、64或71所示的重链可变区序列上具有1-10个氨基酸变化。
在一些实施方式中，所述变体是在SEQ ID NO:45、51、56、64或71所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自：
在SEQ ID NO:45所示的重链可变区上的N84S、S85R和其任意组合的氨基酸 回复突变；
或在SEQ ID NO:51所示的重链可变区上的M48I、R72V、V79A和其任意组合的氨基酸回复突变；
或在SEQ ID NO:56所示的重链可变区上的Y27F、M48I、R72V、V79A、S84N和其任意组合的氨基酸回复突变；
或在SEQ ID NO:64所示的重链可变区上的R38K、R67K、R72V、T74K、M48I、V68A、M70L、V79A和其任意组合的氨基酸回复突变；
或在SEQ ID NO:71所示的重链可变区上的G27Y、M48I、L83F、A97T和其任意组合的氨基酸回复突变。
在一些实施方式中，其中所述人源化抗体包含选自：(vi)SEQ ID NO:45或50所示的重链可变区；
(vii)SEQ ID NO:51、54至55中任一个所示的重链可变区；
viii)SEQ ID NO:56、61、62和63中任一个所示的重链可变区；
ix)SEQ ID NO:64、67、68、69和70中任一个所示的重链可变区；和
x)SEQ ID NO:71、75、76和77中任一个所示的重链可变区。
在一些实施方式中，其中所述的人源化抗体含有SEQ ID NO:46、52、57、65或72所示的轻链可变区或其变体；所述回复突变是在SEQ ID NO:46、52、57、65或72所示的轻链可变区上具有1-10个氨基酸变化的序列。
在一些实施方式中，其中所述变体是在SEQ ID NO:46、52、57、65或72所示的轻链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自：
在SEQ ID NO:46所示的轻链可变区上的S60D、T85D、A43S、S63T和其任意组合的氨基酸回复突变；
或在SEQ ID NO:52所示的轻链可变区上的A43S的氨基酸回复突变；
或在SEQ ID NO:57所示的轻链可变区上的Q3V、A43S、S60D、Y87F和其任意组合的氨基酸回复突变；
或在SEQ ID NO:65所示的轻链可变区上的A43S、I48V和其任意组合的氨基酸回复突变；
或在SEQ ID NO:72所示的轻链可变区上的N22S、P49S和其任意组合的氨基酸回复突变。
在一些实施方式中，其中所述人源化抗体包含选自：
xi)SEQ ID NO:46、47、48和49中任一个所示的轻链可变区；
xii)SEQ ID NO:52或53所示的轻链可变区；
xiii)SEQ ID NO:57、58、59和60中任一个所示的轻链可变区；
xiv)SEQ ID NO:65或66所示的轻链可变区；或
xv)SEQ ID NO:72、73和74中任一个所示的轻链可变区。
在一些实施方式中，其中所述人源化抗体包含选自：
xvi)SEQ ID NO:45或50所示的重链可变区和SEQ ID NO:46、47、48和49中任一个所示的轻链可变区；
xvii)SEQ ID NO:51，54和55中任一个所示的重链可变区和SEQ ID NO:52或53所示的轻链可变区；
xviii)SEQ ID NO:56、61、62和63中任一个所示的重链可变区和SEQ ID NO:57、58、59和60中任一个所示的轻链可变区；
xix)SEQ ID NO:64、67、68、69和70中任一个所示的重链可变区和SEQ ID NO:65或66所示的轻链可变区；或
xx)SEQ ID NO:71、75、76和77中任一个所示的重链可变区和SEQ ID NO:72、73和74中任一个所示的轻链可变区。
在一些实施方式中，单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，其中重链恒定区优选人IgG1、IgG2、IgG3和IgG4抗体重链恒定区，更优选包含SEQ ID NO:78所示的人抗体重链恒定区和SEQ ID NO:79所示的人轻链恒定区。
在一些实施方式中，单克隆抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
在一些实施方式中，单克隆抗体或其抗原结合片段，其与前述的单克隆抗体或其抗原结合片段竞争结合人TIGIT。
另一方面，本公开提供一种药物组合物，其含有治疗有效量的根据上述单克隆抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。药物组合物单位剂量中含单克隆抗体或其抗原结合片段的量优选为0.1-2000mg，更优选为0.1-1000mg。
另一方面，本公开提供一种分离的核酸分子，其编码上述单克隆抗体或其抗原结合片段。
另一方面，本公开提供一种重组载体，其包含上述核酸分子。
另一方面，本公开提供一种用根据上述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
另一方面，本公开提供用于生产上述单克隆抗体或其抗原结合片段的方法，所述方法包括将上述宿主细胞在培养物中进行培养以形成并积累上述单克隆抗体或其抗原结合片段，以及从培养物回收所述单克隆抗体或其抗原结合片段。
另一方面，本公开提供用于检测或测定人TIGIT的方法，所述方法包括使用上述单克隆抗体或其抗原结合片段的步骤。
另一方面，本公开提供用于检测或测定人TIGIT的试剂，所述试剂包含上述单克隆抗体或其抗原结合片段。
另一方面，本公开提供用于治疗与人TIGIT相关的疾病的试剂，所述治疗剂包含上述单克隆抗体或其抗原结合片段，或包含上述药物组合物，或包含上述的核酸分子，所述疾病优选是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，更优选是肿瘤、癌症、免疫性疾病或感染性病症。其中癌症优选非小细胞肺癌，小细胞肺癌，肾细胞癌，结直肠癌，卵巢癌，乳腺癌，胰腺癌，胃癌，膀胱癌，食道癌，间皮瘤，黑素瘤，头和颈癌，甲状腺癌，肉瘤，前列腺癌，成胶质细胞瘤，宫颈癌，胸腺癌，白血病，淋巴瘤，骨髓瘤，蕈样肉芽肿、梅克尔细胞癌、肾上腺皮质癌、肝脏肝细胞癌、胰管腺癌、嗜铬细胞瘤和神经节细胞瘤、子宫内膜癌和卵巢浆液性囊腺癌。其中骨髓瘤优选多发性骨髓瘤(MM)。免疫性疾病优选关节炎、炎性肠病、银屑病。感染性疾病优选慢性病毒感染。另一方面，本公开提供治疗与人TIGIT相关的疾病的方法，所述方法包括向受试者施用药物有效量的上述单克隆抗体或其抗原结合片段，或包含上述的药物组合物，或上述的核酸分子，以治疗人TIGIT相关的疾病，所述疾病优选是T细胞功能障碍性病症，更优选是肿瘤、癌症或感染性病症，最优选CD155阳性或PVR阳性的肿瘤、癌症、或免疫性疾病或感染性病症。
另一方面，本公开提供上述单克隆抗体或其抗原结合片段，或包含上述的药物组合物，或上述核酸分子在制备与人TIGIT相关的疾病的治疗剂中的应用，所述疾病优选是T细胞功能障碍性病症，更优选是肿瘤、癌症或感染性病症，最优选CD155阳性或PVR阳性的肿瘤、癌症、免疫性疾病或感染性病症。
另一方面，本公开提供一种治疗疾病的方法，所述方法包括向受试者施用药物有效量的上述单克隆抗体或其抗原结合片段，或包含上述药物组合物，或上述分离的核酸分子，所述疾病优选是T细胞功能障碍性病症，更优选是肿瘤、癌症或感染性病症，最优选CD155阳性或PVR阳性的肿瘤、癌症或感染性病症。
附图说明
图1：TIGIT抗体结合人TIGIT蛋白的ELISA检测。
图2：TIGIT抗体结合猴TIGIT蛋白的ELISA检测。
图3：TIGIT抗体与过表达人TIGIT的CHO细胞的结合检测。
图4：TIGIT抗体与人PBMC的结合亲和力检测。
图5A：ch1708及其人源化抗体对人TIGIT与CD155结合的阻断作用检测；图5B：TIGIT人源化抗体对人TIGIT与CD155结合的阻断作用检测。
图6：TIGIT抗体对TIGIT抗原与过表达CD155的CHO细胞结合的阻断实验。
图7：TIGIT抗体对CD155与过表达TIGIT的CHO细胞结合的阻断实验。
图8：TIGIT抗体对TIGIT抗原与过表达CD112的CHO细胞结合的阻断实验。
图9：TIGIT抗体在TIGIT过表达的CHO细胞中的结合内吞实验，其中内吞 1小时。
图10A：TIGIT抗体促进自然杀伤细胞(NK)的细胞杀伤实验；图10B：TIGIT抗体促进自然杀伤细胞(NK)的细胞杀伤实验。
图11：TIGIT抗体对PBMC-T淋巴细胞激活实验。
图12：TIGIT人源化抗体在大鼠体内的药代动力学检测。
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
在本公开中，本公开所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本公开中，本公开所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人TIGIT的单克隆抗体。制备时用TIGIT抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源TIGIT抗 体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的TIGIT嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的TIGIT嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(如YTE突变或回复突变、S228P)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，包括CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本公开一个优选的实施方案中，所述的TIGIT人源化抗体中鼠的CDR序列选自SEQ ID NO:15-44；人的抗体可变区框架经过设计选择，其中所述抗体重链可变区上的重链FR区序列，人种系重链选自：(IGHV3-7*01和hjh2)，(IGHV1-46*01和hjh4.1)和(IGHV1-69*02和hjh4.1)的组合序列，和人种系轻链选自：(IGKV1-39*02和hjk2.1)，(IGKV1-39*01和hjk4.1)和(IGKV4-1*01和hjk4.1)的组合序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变(回复突变，即将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基)，以保持活性。
CDR的移植可由于与抗原接触的构架残基而导致产生的TIGIT抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人TIGIT抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查鼠单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高 相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在类似“具有3，2或1个氨基酸差异的变体”中“氨基酸差异”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个或数个氨基酸的插入、缺失或替换。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，TIGIT)的能力的一个或多个片段。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
本公开的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本公开的Fab可以通过用木瓜蛋白酶处理本公开的特异性识别人TIGIT并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
本公开的F(ab')2可以通过用胃蛋白酶处理本公开的特异性识别人TIGIT并与 胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别TIGIT并与胞外区的氨基酸序列或其三维结构结合的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得本公开的特异性识别人TIGIT并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别人TIGIT并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得本公开的特异性识别人TIGIT并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
包含CDR的肽是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含 多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本公开的包含CDR的肽可以通过以下步骤来生产：构建本公开的特异性识别人TIGIT并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
本文中使用的术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，TIGIT分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语"KD"或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10-7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合TIGIT，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如 TIGIT 抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化 抗原(所述抗原在固态表面或细胞表面)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人TIGIT或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码 重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人TIGIT特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、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，其中所述流体与细胞接触。“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例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，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，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床有测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而 变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
此外，本公开包括用于治疗与TIGIT相关的疾病的药剂，所述药剂包含本公开 的单克隆抗体或其抗体片段作为活性成分。
对与TIGIT相关的疾病没有限制，只要它是与TIGIT相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类TIGIT然后阻遏或抑制T细胞功能障碍性病症，优选是恶性肿瘤、癌症或感染性病症，优选在靶向免疫治疗检查点的免疫治疗药物的临床试验中观察到的有临床应答的肿瘤或癌症类型，最优选CD155阳性的肿瘤、癌症或感染性病症。
此外，本公开涉及用于免疫检测或测定TIGIT的方法、用于免疫检测或测定TIGIT的试剂、用于免疫检测或测定表达TIGIT的细胞的方法和用于诊断与TIGIT相关的疾病的诊断剂，其包含本公开的特异性识别人TIGIT并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定TIGIT的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与TIGIT相关的疾病可以通过用本公开的单克隆抗体或抗体片段检测或测定表达TIGIT的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定TIGIT的活体样品没有特别限制，只要它具有包含表达TIGIT的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
本公开实施例提供的TIGIT单克隆抗体或抗原结合片段对TIGIT具有很高的特异性、以及与TIGIT的高亲和力，其中人源化抗体的免疫原性大大降低，同时完全保留了鼠源抗体的特异性，较高的亲和力和优异的体内外活性。
本公开实施例提供的TIGIT单克隆抗体或抗原结合片段具有良好的大鼠上的代谢动力学特性，显示出很长的半衰期，很高的生物利用度。
本公开实施例提供的TIGIT人源化抗体分子具有良好的长期稳定性，无明显异常化学修饰，高浓度下无明显聚集，有较高的纯度和热稳定性。
本公开实施例提供的TIGIT单克隆抗体或抗原结合片段具有良好的增强NK细胞和T细胞活性的效果。
具体实施方式
实施例与测试例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1、TIGIT抗原抗体的制备
1.1蛋白设计及表达
以人TIGIT蛋白(Uniprot号：Q495A1)作为本公开TIGIT的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列，可选的在TIGIT蛋白基础上融合不同的标签，分别克隆到pHr载体上(自产)或pXC-17.4载体上(LONZA)上，在293细胞瞬转表达或CHO细胞稳定表达纯化，获得编码本公开抗原及检测用蛋白。以下TIGIT抗原未特殊说明的均指人TIGIT。
TIGIT胞外区与小鼠IgG2aFc段的融合蛋白：TIGIT-mFc，用于免疫和检测
Figure PCTCN2018108246-appb-000002
注释：划横线部分为信号肽，斜体部分为mFc。
TIGIT胞外区与人IgG1 Fc段的融合蛋白：TIGIT-Fc，用于检测
Figure PCTCN2018108246-appb-000003
Figure PCTCN2018108246-appb-000004
注释：划横线部分为信号肽，斜体部分为Fc。
全长TIGIT：用于构建TIGIT过表达细胞株，用于检测
Figure PCTCN2018108246-appb-000005
信号肽(单下划线)+胞外区+
Figure PCTCN2018108246-appb-000006
下划线
Figure PCTCN2018108246-appb-000007
+胞内区(斜体部分)
cynoTIGIT胞外区与小鼠IgG2aFc段的融合蛋白：cynoTIGIT-mFc，用于检测
Figure PCTCN2018108246-appb-000008
注释：划横线部分为信号肽，斜体部分为mFc。
1.2 TIGIT相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
(1)杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清纯化首选ProteinG进行亲和层析，将培养所得杂交瘤离心取上清，根据上清体积加入10-15％体积的1M Tris-HCl(pH8.0-8.5)调节上清pH。ProteinG柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M醋酸/醋酸钠(pH3.0)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者 利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
(2)Protein A亲和层析提取带Fc标签的融合蛋白或者抗体：
首先将表达Fc融合蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
实施例2、抗人TIGIT杂交瘤单克隆抗体的制备
2.1免疫
抗人TIGIT单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为带mFc的人TIGIT胞外区(SEQ ID NO:1)。
免疫方案：用
Figure PCTCN2018108246-appb-000009
Gold Adjuvant(Sigma Cat No.T2684)与Thermo
Figure PCTCN2018108246-appb-000010
Alum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂(
Figure PCTCN2018108246-appb-000011
Gold Adjuvant)比例为1:1，抗原与佐剂(Thermo
Figure PCTCN2018108246-appb-000012
Alum)比例为3:1，50μg/只/次(首次免疫)，25μg/只/次(加强免疫)。抗原乳化后进行接种，时间为第0、14、28、42、56天。第0天腹膜内(IP)注射50μg/只的乳化后抗原。第14天皮下(sc)多点(一般背部6-8点)注射25μg/只。第28，42天根据背部结块和腹部肿胀情况，选择背部或腹膜内注射抗原。于第21，35，49，63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
2.2脾细胞融合
采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018108246-appb-000013
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以0.5-1×10^6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的DMEM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全 培养基100μl/孔，继续培养3-4天至形成针尖般克隆。去除上清，加入200μl/well的HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
2.3杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测。并将结合ELISA检测的阳性孔细胞上清进行细胞结合实验和细胞阻断实验。结合和阻断均为阳性的孔细胞及时进行扩增冻存保种和二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞也均需进行TIGIT结合ELISA、HTRF阻断实验、细胞结合实验和细胞阻断实验检测。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
2.4杂交瘤阳性克隆序列测定
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TMReverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增后测序。从得到的DNA序列获得所筛选到的阳性克隆m17对应的抗体可变区氨基酸序列如下所示：
m1707-HCVR
Figure PCTCN2018108246-appb-000014
m1707-LCVR
Figure PCTCN2018108246-appb-000015
m1708-HCVR
Figure PCTCN2018108246-appb-000016
m1708-LCVR
Figure PCTCN2018108246-appb-000017
m1709-HCVR
Figure PCTCN2018108246-appb-000018
m1709-LCVR
Figure PCTCN2018108246-appb-000019
m1710-HCVR
Figure PCTCN2018108246-appb-000020
m1710-LCVR
Figure PCTCN2018108246-appb-000021
m1711-HCVR
Figure PCTCN2018108246-appb-000022
m1711-LCVR
其中各抗体轻重链中CDR序列如表1所示。
表1各重链及轻链CDR区序列
Figure PCTCN2018108246-appb-000024
Figure PCTCN2018108246-appb-000025
实施例3、鼠源抗人TIGIT抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中关键氨基酸回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，即得到人源化抗TIGIT单克隆抗体。其中CDR区氨基酸残基的确定由Kabat编号系统确定并注释。
上述鼠源抗体的轻重链可变区与人源抗体的轻重链恒定区连接后形成嵌合抗体，m1707抗体对应的嵌合抗体命名为ch1707，其他抗体类推。
3.1杂交瘤克隆m1707的人源化
(1)m1707人源化构架选择
鼠源抗体m1707的人源化轻链模板为IGKV1-39*02和hjk2.1，人源化重链模板为IGHV3-7*01和hjh2，经人源化后得到人源化抗体h1707，人源化可变区序列如下：
h1707 VH-CDR graft
Figure PCTCN2018108246-appb-000026
h1707VL-CDR graft
Figure PCTCN2018108246-appb-000027
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1707回复突变设计如下表：
Figure PCTCN2018108246-appb-000028
注：如S60D表示依照氨基酸序列自然顺序编号，将60位S突变回D。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1707人源化序列组合如下表：
  h1707-H1 h1707-H2
h1707-L1 h1707-01 h1707-02
h1707-L2 h1707-03 h1707-04
h1707-L3 h1707-05 h1707-06
h1707-L4 h1707-07 h1707-08
注：该表表示各种突变组合所得的序列。如h1707-04表示，在人源化的抗体h1707-04上有轻链L2、重链H2的四个回复突变。其它类推。
(4)h1707人源化具体序列如下：
>h1707-L1(同h1707 VL-CDR graft)
Figure PCTCN2018108246-appb-000029
>h1707-L2
Figure PCTCN2018108246-appb-000030
>h1707-L3
Figure PCTCN2018108246-appb-000031
>h1707-L4
Figure PCTCN2018108246-appb-000032
>h1707-H1(同h1707 VH-CDR graft)
Figure PCTCN2018108246-appb-000033
>h1707-H2
Figure PCTCN2018108246-appb-000034
3.2杂交瘤克隆m1708的人源化
(1)m1708人源化构架选择
鼠源抗体m1708的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV1-46*01和hjh4.1，经人源化后得到人源化抗体h1708，人源化可变区序 列如下：
h1708VH-CDR graft
Figure PCTCN2018108246-appb-000035
h1708VL-CDR graft
Figure PCTCN2018108246-appb-000036
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1708回复突变设计如下表：
Figure PCTCN2018108246-appb-000037
注：如A43S表示依照氨基酸序列自然顺序编号，将43位A突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1708人源化序列组合如下表：
  h1708-H1 h1708-H2 h1708-H3
h1708-L1 h1708-01 h1708-02 h1708-03
h1708-L2 h1708-04 h1708-05 h1708-06
注：该表表示各种突变组合所得的序列。如h1708-05表示，在人源化的抗体h1708-05上有轻链L2、重链H2的两个回复突变。其它类推。
(4)h1708人源化具体序列如下：
>h1708-L1(同h1708 VL-CDR graft)
Figure PCTCN2018108246-appb-000038
>h1708-L2
Figure PCTCN2018108246-appb-000039
>h1708-H1(同h1708 VH-CDR graft)
Figure PCTCN2018108246-appb-000040
>h1708-H2
Figure PCTCN2018108246-appb-000041
>h1708-H3
Figure PCTCN2018108246-appb-000042
3.3杂交瘤克隆m1709的人源化
(1)m1709人源化构架选择
鼠源抗体m1709的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV1-46*01和hjh4.1，经人源化后得到人源化抗体h1709，人源化可变区序列如下：
h1709VH-CDR graft
Figure PCTCN2018108246-appb-000043
h1709VL-CDR graft
Figure PCTCN2018108246-appb-000044
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1709回复突变设计如下表：
Figure PCTCN2018108246-appb-000045
注：如S60D表示依照氨基酸序列自然顺序编号，将60位S突变回A。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1709人源化序列组合如下表：
  h1709-H1 h1709-H2 h1709-H3 h1709-H4
h1709-L1 h1709-01 h1709-02 h1709-03 h1709-04
h1709-L2 h1709-05 h1709-06 h1709-07 h1709-08
h1709-L3 h1709-09 h1709-10 h1709-11 h1709-12
h1709-L4 h1709-13 h1709-14 h1709-15 h1709-16
注：该表表示各种突变组合所得的序列。如h1709-06表示，在人源化的抗体h1709-06上有轻链L2、重链H2的三个回复突变。其它类推。
(4)h1709人源化具体序列如下：
>h1709-L1(同h1709 VL-CDR graft)
Figure PCTCN2018108246-appb-000046
>h1709-L2
Figure PCTCN2018108246-appb-000047
>h1709L3
Figure PCTCN2018108246-appb-000048
Figure PCTCN2018108246-appb-000049
>h1709-L4
Figure PCTCN2018108246-appb-000050
>h1709.H1(同h1709 VH-CDR graft)
Figure PCTCN2018108246-appb-000051
>h1709-H2
Figure PCTCN2018108246-appb-000052
>h1709-H3
Figure PCTCN2018108246-appb-000053
>h1709-H4
Figure PCTCN2018108246-appb-000054
3.4杂交瘤克隆m1710的人源化
(1)m1710人源化构架选择
鼠源抗体m1710的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV1-46*01和hjh4.1，经人源化后得到人源化抗体h1710，人源化可变区序列如下：
h1710VH-CDR graft
Figure PCTCN2018108246-appb-000055
Figure PCTCN2018108246-appb-000056
h1710VL-CDR graft
Figure PCTCN2018108246-appb-000057
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1710回复突变设计如下表：
Figure PCTCN2018108246-appb-000058
注：如A43S表示依照氨基酸序列自然顺序编号，将43位A突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1710人源化序列组合如下表：
  h1710-H1 h1710-H2 h1710-H3 h1710-H4 h1710-H5
h1710-L1 h1710-01 h1710-02 h1710-03 h1710-04 h1710-05
h1710-L2 h1710-06 h1710-07 h1710-08 h1710-09 h1710-10
注：该表表示各种突变组合所得的序列。如h1710-07表示，在人源化的抗体h1710-07上有轻链L2、重链H2的四个回复突变。其它类推。
(4)h1710人源化具体序列如下：
>h1710-L1(同h1710 VL-CDR graft)
Figure PCTCN2018108246-appb-000059
>h1710-L2
Figure PCTCN2018108246-appb-000060
>h1710-H1(同h1710 VH-CDR graft)
Figure PCTCN2018108246-appb-000061
>h1710-H2
Figure PCTCN2018108246-appb-000062
>h1710-H3
Figure PCTCN2018108246-appb-000063
>h1710-H4
Figure PCTCN2018108246-appb-000064
>h1710-H5
Figure PCTCN2018108246-appb-000065
3.5杂交瘤克隆m1711的人源化
(1)m1711人源化构架选择
鼠源抗体m1711的人源化轻链模板为IGKV4-1*01和hjk4.1，人源化重链模板为IGHV1-69*02和hjh4.1，经人源化后得到人源化抗体h1711，人源化可变区序列如下：
h1711VH-CDR graft
Figure PCTCN2018108246-appb-000066
Figure PCTCN2018108246-appb-000067
h1711VL-CDR graft
Figure PCTCN2018108246-appb-000068
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1711回复突变设计如下表：
Figure PCTCN2018108246-appb-000069
注：如P49S表示依照氨基酸序列自然顺序编号，将49位P突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1711人源化序列组合如下表：
  h1711-H1 h1711-H2 h1711-H3 h1711-H4
h1711-L1 h1711-01 h1711-02 h1711-03 h1711-04
h1711-L2 h1711-05 h1711-06 h1711-07 h1711-08
h1711-L3 h1711-09 h1711-10 h1711-11 h1711-12
注：该表表示各种突变组合所得的序列。如h1711-06表示，在人源化的抗体h1711-06上由轻链L2、重链H2组合成的回复突变抗体。其它类推。
(4)h1711人源化具体序列如下：
>h1711-L1(同h1711 VL-CDR graft)
Figure PCTCN2018108246-appb-000070
>h1711-L2
Figure PCTCN2018108246-appb-000071
>h1711-L3
Figure PCTCN2018108246-appb-000072
>h1711-H1(同h1711 VH-CDR graft)
Figure PCTCN2018108246-appb-000073
>h1711-H2
Figure PCTCN2018108246-appb-000074
>h1711-H3
Figure PCTCN2018108246-appb-000075
>h1711-H4
Figure PCTCN2018108246-appb-000076
以上各重链可变区可与如SEQ ID NO:78所示的重链恒定区序列(IgG4，带S228P突变)重组表达得到最终的完整重链序列。上述各轻链可变区与如SEQ ID NO:79所示的轻链恒定区序列(kappa链)重组表达得到最终的完整重轻链序列。上述重轻链可变区也可与其他本领域内公知的IgG家族的重轻链恒定区或突变的IgG家族恒定区重组，形成完整的抗体重轻链序列。示例性的恒定区序列如下所示：
IgG4重链恒定区，带S228P突变：
Figure PCTCN2018108246-appb-000077
轻链恒定区：
Figure PCTCN2018108246-appb-000078
采用所属领域的常规技术手段，将以上轻链、重链与本公开技术方案可选的人源化恒定区，及经过功能性修修饰的人源化恒定区，重组并表达。
阳性对照抗体为22G2-H3Q，VH和VL序列来自US20160176963A1(分别为US20160176963A1的SEQ ID NO：8和9)，分别与SEQ ID NO：78和SEQ ID NO：79所示的重链恒定区和轻链恒定区形成完整全长抗体。22G2-H3Q的VH和VL具体序列如下：
22G2-H3Q VH
Figure PCTCN2018108246-appb-000079
22G2-H3Q VL
Figure PCTCN2018108246-appb-000080
以下用生化测试方法验证本公开的结合活性
测试例1：TIGIT抗体结合人TIGIT蛋白的ELISA实验
抗TIGIT抗体的结合力通过抗体与人TIGIT蛋白的ELISA实验来检测。用带Fc或mFc标签的TIGIT融合蛋白通过与包被在酶标板中的抗Fc或mFc抗体结合从而固定到96孔酶标板中，抗体加入后信号的强弱被用于判断抗体和TIGIT的结合活性，阳性对照分子为10A7 hIgG4(其中10A7 IgG4的轻重链可变区序列来自 US20130251720A1的SEQ ID NO：21和22，与上述轻重链可变区连接形成全长抗体的轻重链恒定区分别为本申请的SEQ ID NO：79和78)，阴性对照为与TIGIT不结合的人IgG4抗体(hIgG4)，具体实验方法如下：
用pH7.4的PBS(上海源培，Cat No.B320)缓冲液将羊抗人Fc抗体(Jackson Immuno Research，Cat No.109-005-008)或羊抗鼠Fc抗体(Sigma，Cat No.M3534-1ML)稀释至2μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat No.232100)封闭液200μl/孔，37℃孵育箱孵育3小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.05％tween-20)洗板5次后，加入50μl/孔用样品稀释液(pH7.4 PBS含1％BSA)稀释至0.5μg/ml的TIGIT-Fc融合蛋白(内部生产)或TIGIT-mFc融合蛋白(内部生产)，置37℃孵育箱孵育1小时或4℃放置过夜。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板5次，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用酶标仪(Thermo scientific Multiskan,MK3)在波长450nm处读取吸收值，用GraphPad Prism 5分析数据，计算TIGIT抗体对人TIGIT蛋白的结合EC50值。结果见图1。
测试例2：TIGIT抗体结合食蟹猴TIGIT蛋白的ELISA实验
抗TIGIT抗体的猴交叉结合力通过抗体与食蟹猴(cynomolgus)TIGIT蛋白的ELISA实验来检测。用带Fc或mFc标签的食蟹猴TIGIT融合蛋白通过与包被在酶标板中的抗Fc或mFc抗体结合从而固定到96孔酶标板中，抗体加入后信号的强弱被用于判断抗体和食蟹猴TIGIT的结合活性，具体实验方法如下：
用pH7.4的PBS(上海源培，Cat No.B320)缓冲液将羊抗人Fc抗体(Jackson Immuno Research，Cat No.109-005-008)或羊抗鼠Fc抗体(Sigma，Cat No.M3534-1ML)稀释至2μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat No.232100)封闭液200μl/孔，37℃孵育箱孵育3小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液(pH7.4 PBS含1％BSA)稀释至0.5μg/ml的食蟹猴TIGIT-Fc融合蛋白(内部生产)或食蟹猴TIGIT-mFc融合蛋白(内部生产)，置37℃孵育箱孵育1小时或4℃放置过夜。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次 后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板5次，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用酶标仪(Thermo scientific Multiskan MK3)在波长450nm处读取吸收值，用GraphPad Prism 5分析数据，计算TIGIT抗体对猴TIGIT的结合EC50值。结果见图2。
测试例3：TIGIT抗体与人TIGIT过表达CHO细胞的结合实验
抗TIGIT抗体的结合力通过抗体与过表达TIGIT蛋白的CHO细胞的结合实验来检测。通过电转染的方法将TIGIT全长质粒转进CHO细胞中加压筛选两周后，检测TIGIT的表达量。将过表达细胞固定于96孔板底后，抗体加入后信号的强弱被用于判断抗体和TIGIT过表达CHO细胞的结合活性，具体实验方法如下：
将细胞以5×10^5/ml密度，100μl/孔接种于96孔板中过夜培养。弃上清，用PBS洗三遍后，加入100μl/孔的细胞免疫固定液(Beyotime，Cat No.P0098)室温固定半小时，PBS洗四遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat No.232100)封闭液200μl/孔，37℃孵育箱孵育3小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板5次，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H2SO4终止反应，用酶标仪(Thermo scientific Multiskan MK3)在波长450nm处读取吸收值，用GraphPad Prism 5分析数据，计算TIGIT抗体对TIGIT过表达CHO细胞的结合EC50值。结果见图3及下表。
Figure PCTCN2018108246-appb-000081
测试例4：TIGIT抗体与人PBMC的结合实验
抗TIGIT抗体的结合力通过抗体与体外激活的人PBMC的结合实验来检测。通过超抗原金黄色葡萄球菌肠毒素B(SEB)刺激的方法使人PBMC活化，抗体加入后荧光信号的强弱被用于判断抗体和活化的人PBMC的结合活性，具体实验 方法如下：
新鲜血液利用Ficoll-Hypaque密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI 1640培养基中培养，该培养基中添加10％(v/v)FBS，同时加入超抗原金黄色葡萄球菌肠毒素B(SEB)500ng/ml，37℃、5％CO 2条件下培养4天。
激活后的PBMC细胞以5×10^6/ml密度，100μl/孔接种于96孔圆底板(Corning，Cat No.32915001)中，离心机(Beckman Coulter,Allegra X-15R Centrifuge)1500rpm离心5分钟，弃上清；用200μl PBS重悬细胞，离心，弃上清，重复一遍。加入100μl/孔已用样品稀释液(pH7.4PBS含1％BSA)梯度稀释的待测抗体溶液重悬细胞，4℃孵育1小时。孵育结束后，1500rpm离心5分钟，弃上清，用样品稀释液洗两遍细胞后，加入PE-山羊抗人IgG(Jackson ImmunoResearch，109-115-098)稀释液100μl重悬细胞，4℃孵育1小时。孵育结束后，1500rpm离心5分钟，弃上清，用样品稀释液洗两遍细胞，最后用200μl/孔样品稀释液重悬细胞，在流式细胞仪(BD FACS CantoⅡ)上检测荧光信号强弱，用GraphPad Prism 5分析数据，计算TIGIT抗体对人PBMC细胞结合的EC50值。结果见图4。
测试例5：Biacore测定
用Biacore，GE仪器测定待测人源化抗TIGIT抗体与人、猴TIGIT的亲和力。
用Protein A生物传感芯片(Cat.#29127556,GE)亲和捕获或按照人抗捕获试剂盒(Cat.#28-9538-28，GE)说明书中的方法将人抗捕获抗体共价偶联于生物传感芯片(Cat.#28-9538-28，GE)上，从而亲和捕获一定量的待测抗体，然后于芯片表面流经一系列浓度梯度下的人、猴TIGIT抗原，人源TIGIT可选自义翘神州生物公司(Cat.10917-H08-H，Sino.Biol)，猴TIGIT由实施例1及实施例2表达纯化而得，利用Biacore仪器(Biacore T200，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液或pH1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54,GE)将生物芯片洗净再生。实验中用到的氨基偶联试剂盒购自GE公司(Cat.#BR-1000-50，GE)，缓冲液为HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1×(pH 7.4)。
实验得到的数据用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型进行拟合，得出亲和力数值，结果见表2-4。
表2、待测分子与huTIGIT蛋白的反应亲和力
Figure PCTCN2018108246-appb-000082
Figure PCTCN2018108246-appb-000083
表3、待测分子与cynoTIGIT蛋白的反应亲和力
Figure PCTCN2018108246-appb-000084
表4、ch1711及其人源化抗体与TIGIT蛋白的反应亲和力
Figure PCTCN2018108246-appb-000085
测试例6：TIGIT抗体对TIGIT抗原与CD155蛋白结合的阻断试验
抗TIGIT抗体的阻断能力通过抗体阻断TIGIT与CD155蛋白结合的HTRF实验来检测。用Pab Anti-Human IgG-Tb(Cisbio，Cat No.61HFCTAA)和Streptavidin-XL665(Cisbio，Cat No.610SAXLA)这对供体和受体与TIGIT-Fc和biotinylated CD155(R&D，Cat No.2530-CD-050/CF)结合，或者Pab Anti-mouse-IgG-XL665(Cisbio，Cat No.61PAMXLA)和Streptavidin-Tb(Cisbio，Cat No.610SATLA)与TIGIT-mFc和biotinylated CD155(R&D，Cat No.2530-CD-050/CF)结合，杂交瘤纯化抗体或人源化抗体加入后信号的强弱被用于判断抗体阻断TIGIT与CD155的活性，具体实验方法如下：
将稀释液(pH7.4 PBS含1％BSA)稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)10μl/孔加入384孔实验板(Corning,Cat No.3706)，1000rpm离心1min后加入2.5μl/孔用样品稀释液稀释至2μg/ml的TIGIT-Fc或TIGIT-mFc，1000rpm离心1min后加入2.5μl/孔稀释至4μg/ml的biotin-CD155，1000rpm离心1min后室温预孵育10min，然后加入2.5μl/孔的样品稀释液稀释至3.2μg/ml的Pab  Anti-Human IgG-Tb(Cisbio，Cat No.61HFCTAA)和2.5μl/孔的0.08μg/ml的Streptavidin-XL665(Cisbio，Cat No.610SAXLA)，或者2.5μl/孔的样品稀释液稀释至3.2μg/ml的Pab Anti-mouse-IgG-XL665(Cisbio，Cat No.61PAMXLA)和2.5μl/孔的0.08μg/ml的Streptavidin-Tb(Cisbio，Cat No.610SATLA)，室温放置1小时，用PHEARstar FS酶标仪(BMG LABTECH)检测665nm与620nm的发射光值，用GraphPad Prism 5分析数据，计算TIGIT抗体对人TIGIT与CD155蛋白的抑制活性。结果见图5A和图5B。
测试例7：TIGIT抗体对TIGIT抗原与过表达CD155的CHO细胞结合的阻断实验
抗TIGIT抗体的阻断能力通过抗体阻断TIGIT与过表达CD155的CHO细胞结合的ELISA实验来检测。通过电转染的方法将CD155全长质粒转染进CHO细胞中加压筛选两周后，检测CD155的表达量。将过表达细胞固定于96孔板底后，TIGIT与稀释好的不同浓度抗TIGIT抗体预孵育后加入孔板，加入二抗后通过信号的强弱被用于判断抗体阻断TIGIT与过表达CD155的CHO细胞结合的能力，具体实验方法如下：
CD155-CHO细胞以5×10^5/ml密度，100μl/孔接种于96孔板中过夜培养。弃上清，用PBS洗三遍后，加入100μl/孔细胞免疫固定液(Beyotime，Cat No.P0098)室温固定半小时，PBS洗四遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat No.232100)封闭液200μl/孔，37℃孵育箱孵育3小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液(pH7.4PBS含1％BSA)稀释的已预混合孵育1小时的终浓度为0.2μg/ml的human TIGIT-hFc(内部生产)或human TIGIT-mFc(内部生产)与梯度浓度的待测抗体的抗原抗体混合液，置37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)或羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15分钟，加入50μl/孔1M H2SO4溶液终止反应，用酶标仪(Thermo scientific Multiskan，MK3)在波长450nm处读取吸收值，用GraphPad Prism 5分析数据，计算TIGIT抗体对抗原与过表达CD155的CHO细胞结合的阻断作用。结果见图6。
测试例8：TIGIT抗体对CD155蛋白与过表达TIGIT的CHO细胞结合的阻断实验
抗TIGIT抗体的阻断能力通过抗体阻断CD155与TIGIT过表达CHO细胞结合的FACS实验来检测。通过电转染的方法将TIGIT全长质粒转染进CHO细胞中加压筛选两周后，检测TIGIT的表达量。将过表达细胞与不同浓度的抗TIGIT抗 体预孵育后，再加入荧光标记的CD155-Fc进行孵育，通过信号的强弱用于判断抗体阻断CD155与过表达TIGIT的CHO细胞结合的能力，具体实验方法如下：
先将CD155-Fc(Sino Biological，Cat No.10109-H02H)标记CF TM633(Sigma Aldrich,Cat No.MX633S100)的荧光染料。将CD155-Fc用PBS溶解至浓度为0.5-1mg/ml，加入9倍样品体积的10×Mix-n-Stain Reaction Buffer，混匀；然后加入CF TM633荧光染料，避光室温孵育30分钟后，荧光标记完成。
TIGIT-CHO细胞以5×10^6/ml密度，100μl/孔接种于96孔圆底板(Corning，Cat No.32915001)中，离心机(Beckman Coulter,Allegra X-15R Centrifuge)1500rpm离心5分钟，弃上清；用200μl PBS重悬细胞，离心，弃上清，重复一遍。加入100μl/孔已用样品稀释液(pH7.4PBS含1％BSA)梯度稀释的待测抗体溶液重悬细胞，4℃孵育1小时。孵育结束后，1500rpm离心5分钟，弃上清，用样品稀释液洗两遍细胞后，加入100μl 2μg/ml的CF TM633荧光标记的CD155-Fc溶液重悬细胞，4℃孵育1小时。孵育结束后，1500rpm离心5分钟，弃上清，用样品稀释液洗两遍细胞，最后用200μl/孔样品稀释液重悬细胞，在流式细胞仪(BD FACS CantoⅡ)上检测荧光信号强弱，用GraphPad Prism 5分析数据，计算TIGIT抗体对CD155与TIGIT-CHO细胞结合的阻断能力。结果见图7及下表。
Figure PCTCN2018108246-appb-000086
本测试例中h1707-02、h1708-04、h1709-10、h1710-01、h1711-04抗体均能够很好的阻断CD155与TIGIT过表达的CHO细胞的结合。
测试例9：TIGIT抗体对TIGIT抗原与过表达CD112的CHO细胞结合的阻断实验
抗TIGIT抗体的阻断能力通过抗体阻断TIGIT与CD112过表达CHO细胞结合的FACS实验来检测。通过电转染的方法将CD112全长质粒转染进CHO细胞中加压筛选两周后，检测CD112的表达量。将TIGIT-mFc蛋白与稀释好的不同浓度抗TIGIT抗体预孵育后加入CD112过表达的CHO细胞中进行孵育，加入PE label的抗体检测TIGIT信号的强弱被用于判断抗体阻断TIGIT与过表达CD112的CHO细胞结合的能力，具体实验方法如下：
1％BSA稀释人源化抗体样品，从20μg/mL起始浓度两倍稀释九个浓度点，同时稀释TIGIT-mFc至2μg/mL，将抗原和不同浓度的抗体按1:1体积比混匀，37℃预孵育30分钟；收集CD112-CHOs细胞，PBS洗涤一次，按0.5*10^6/test分配；用150μl的抗原抗体混合液重悬细胞，4℃孵育60分钟，1％BSA洗涤3次；PE-山羊抗小鼠IgG(Biolegend,405307)稀释液100μl重悬细胞，4℃孵育40分钟，1％BSA洗涤3次，用200μl 1％BSA重悬细胞，在流式细胞仪BD FACSCantoⅡ上 读取各样品MFI(平均荧光强度)，GraphPad Prism 5分析数据，判断TIGIT抗体对抗原与CD112-CHO细胞结合的阻断作用。结果见图8。
测试例10：TIGIT抗体在TIGIT过表达CHO细胞的结合内吞实验
为了研究TIGIT抗体与细胞表面抗原结合后发生内吞的能力，用过表达全长TIGIT的CHO细胞进行TIGIT抗体的内吞能力的FACS实验。具体实验方法如下：
TIGIT-CHO细胞以2×10^6/ml密度，100μl/孔接种于96孔圆底板(Corning，Cat No.32915001)中，离心机(Beckman Coulter,Allegra X-15R Centrifuge)1500rpm离心5分钟，弃上清；用200μl 1％BSA重悬细胞，离心，弃上清，重复一遍。1％BSA稀释人源化抗体样品至4μg/mL浓度，100μl/孔加入细胞中重悬细胞，冰上孵育1小时。孵育结束后，1500rpm离心5分钟，弃上清，用1％BSA洗三遍细胞后，用10％FBS-DMEM/F-12培养基重悬细胞并将细胞分为两部分，一部分放到37℃培养箱中孵育1小时(内吞组，Internalization group)，另一部分继续在冰上孵育1小时(结合亲和力组，Binding affinity group)。孵育结束后，用1％BSA洗一遍细胞后加入用1％BSA稀释的PE-anti-Fc抗体(Jackson，109-115-098)冰上孵育一小时后，1％BSA洗涤3次，用1％BSA重悬细胞，在流式细胞仪BD FACSCantoⅡ上读取各样品MFI，按照如下计算公式计算抗体的内吞率。注：Black是不加抗TIGIT抗体孵育，只加用1％BSA稀释的PE-抗Fc抗体冰上孵育一小时后洗涤3次重悬读取MFI。结果见图9。
内吞比率(Internalization Ratio)％＝(结合亲和力组-内吞组)*100/(结合亲和力组-空白组)
测试例11：自然杀伤细胞(NK)的细胞杀伤实验
为了研究TIGIT抗体对NK细胞杀伤功能的影响，收集和纯化人外周血单核细胞(PBMC)，提取自然杀伤细胞(NK)，与人大肠癌细胞WiDr共培养4h，检测乳酸脱氢酶(LDH)的分泌水平。具体实验过程如下：
人大肠癌细胞系WiDr于MEM培养基中培养，该培养基中添加10％(v/v)胎牛血清(FBS)，37℃、5％CO 2条件下培养。新鲜血液利用Ficoll-Hypaque密度梯度离心(Stem Cell Technologies)得到PBMC，人原代NK细胞从新鲜分离的PBMC中提取(Miltenyi,CAT#130-092-657)，于RPMI 1640培养基中培养，该培养基中添加10％(v/v)FBS，37℃、5％CO 2条件下培养。
人原代NK细胞接种至6孔细胞培养板，细胞密度约为2×10^6/ml，加入100U/mL人IL-2过夜培养后，利用无酚红RPMI 1640培养基洗涤，重悬，并接种至96孔U底板中，细胞密度约为3×10^5/孔，同时加入梯度稀释的抗体样品(用PBS稀释)或等量的同型IgG作为空白对照。37℃，5％CO 2培养箱孵育1h后，靶标细胞WiDr以1:1比例与人原代NK细胞共培养，37℃，5％CO 2培养箱培养4h后，收集细胞培养上清。采用CytoTox
Figure PCTCN2018108246-appb-000087
Non-Radioactive Cytotoxicity Assay(Promega,CAT#G1780)说明书方法检测细胞培养上清内LDH分泌水平。特异性细 胞溶解的百分比以下式决定：％溶解＝100×(ER-SR1-SR2)/(MR-SR1)，其中ER、SR(1&2)及MR分别代表实验、自发(1为靶标细胞，2为人原代NK细胞)及最大LDH释放。自发释放系由单独培养于培养基中的靶标细胞或人原代NK细胞所释放的LDH，最大释放系利用裂解液裂解所有靶标细胞时所测定的LDH。结果如图10A或图10B以及下表所示，TIGIT人源化候选抗体h1707-02、h1708-04、h1710-01等抗体均能够不同程度增强人原代NK细胞对靶细胞的杀伤，并且有药物浓度剂量效应。
Figure PCTCN2018108246-appb-000088
Figure PCTCN2018108246-appb-000089
测试例12：PBMC-T淋巴细胞激活实验
为了研究TIGIT抗体对人原代T淋巴细胞功能的影响，收集和纯化人外周血单核细胞(PBMC)，采用结核菌素(TB)体外刺激5天后，检测细胞因子IFNγ分泌水平。具体实验过程如下：
新鲜血液利用Ficoll-Hypaque密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI 1640培养基中培养，该培养基中添加10％(v/v)FBS，37℃、5％CO 2条件下培养。
新鲜分离纯化的PBMC以RPMI 1640培养基调整密度为2×10^6/ml，20mL细胞悬液中加入25μl结核菌素，37℃、5％CO 2培养箱培养5天。第5天，在96孔细胞培养板中加入CD155(重组CD155/PVR Protein，R&D，2530-CD-050/CF)，每孔0.25μg，4℃包被过夜。第6天，收集上述培养的细胞离心，用PBS洗一次，重悬至新鲜的RPMI 1640培养基中，调整密度为1×10^6/ml，接种至CD155包被的96孔细胞培养板，每孔90μl。同时加入梯度稀释的抗体样品(用PBS稀释)或等量的同型IgG作为空白对照，每孔10μl。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)收集细胞培养上清，采用ELISA的方法(人IFN-γ检测试剂盒，欣博盛，EHC102g.96)检测IFN-γ的水平。具体操作参考试剂说明书。结果如图11所示，TIGIT人源化候选抗体h1708-04，h1710-01等抗体能够不同程度增强激活的原代T淋巴细胞分泌细胞因子IFN-γ，并且有药物浓度剂量效应。
测试例13：人源化TIGIT抗体大鼠药代动力学评价
SD雄性大鼠，体重180-240g，购自西普尔-必凯实验动物有限公司。饲养期间自由摄取饲料和水，实验室环境适应性饲养不小于3天，12/12小时光/暗周期 调节，温度16-26℃，相对湿度40-70％。实验开始前一天，对SD大鼠进行编号，随机分组，每组3只。实验当天，五组大鼠分别静脉注射或皮下注射受试药物h1707-02、h1708-04、h1710-01、h1711-04和h1709-10，22G2-H3Q为阳性对照，给药剂量为3mg/kg；注射体积均为5ml/kg。
静脉注射给药方式于给药前及给药后5min,8h,1d,2d,4d,7d,10d,14d,21d,28d各时间点采血。皮下注射给药方式于给药前及给药后1h,4h,8h,1d,2d,4d,8d,11d,14d,21d,28d各时间点采血。每只动物取全血0.2ml，不加抗凝剂，取血后在4℃放置30min，1000g离心15min，取上清置于EP管中，-80℃保存。
采用ELISA方法(见实施例3测试例1)检测血清中的抗体浓度，采用Winnolin软件计算受试药物的药动学参数。所得部分主要药动学结果见图12。
经检测，SD大鼠静脉注射给予3mg/kg抗TIGIT抗体h1707-02,h1708-04，h1710-01，h1709-10，h1711-04后，在大鼠体内的暴露量相近；皮下给药生物利用度高，接近100％；各抗体的消除半衰期均较长，优于22G2-H3Q抗体。
测试例14：利用UNIT检测人源化TIGIT抗体的热稳定性
比较了不同的缓冲体系下的热稳定性情况，不同pH对应的示例性缓冲体系如10mM PBS(pH7.4)，15mM His(pH6.0)，10mM Acetate acid(pH5.2)。将样品置换到对应缓冲液中，控制样品浓度在50mg/ml左右，利用UNIT进行检测。检测时，用移液枪吸取9μl样品，加入仪器配置的样品槽中，注意不能有气泡，用夹套将样品槽加紧放入仪器中，设置参数运行：Start Temp 20℃；Incubation0s；Rate 0.3℃/min；Plate Hold 5s；End Temp 95℃。在几个测试体系中h1707-02,h1708-04，h1710-01，h1709-10，h1711-04等抗体均表现了良好的热稳定性。
表5抗体热稳定性监测表
Figure PCTCN2018108246-appb-000090
测试例15、通过SEC-HPLC监测样品纯度考察一定浓度条件下周期稳定性
示例性的条件比如将样品浓度控制在约50mg/ml，在10mM PBS(pH7.4)，15mM His(pH6.0)，10mM Acetate acid(pH5.2)等体系中比较不同抗体在-80℃ 反复冻融5次及4℃和40℃保存28天的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测抗体纯度，h1707-02、h1708-04、h1709-10、h1710-01、h1711-04等均表现了良好的稳定性，一个月40℃加速SEC纯度无明显变化。
Figure PCTCN2018108246-appb-000091
测试例16、抗体的化学稳定性
对h1707-02、h1708-04、h1709-10、h1710-01、h1711-04等抗体分子进行化学稳定性检测。
取500μg待测抗体溶于500μl pH 7.4的PBS中，40℃水浴；分别于0、14、28天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gua-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT，50℃水浴1小时， 后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的trypsin，37℃水浴酶解过夜。Agilent 6530 Q-TOF进行LC-MS检测分析，结果显示h1707-02、h1708-04、h1709-10、h1710-01、h1711-04在40度条件加速一个月均未发现明显的失水、氧化、脱酰胺等不稳定的变化，提示分子良好的化学稳定性。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本公开的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (23)

  1. 一种单克隆抗体或其抗原结合片段，所述单克隆抗体或抗原结合片段特异性结合人TIGIT，所述单克隆抗体包含重链可变区和轻链可变区，其中：
    i)重链可变区包含选自如SEQ ID NO：15、16和17氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：15、16和17所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：18、19和20氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：18、19和20所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
    ii)重链可变区包含选自如SEQ ID NO：21、22和23氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：21、22和23所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：24、25和26氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：24、25和26所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
    iii)重链可变区包含选自如SEQ ID NO：27、28和29氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：27、28和29所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：30、31和32氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：30、31和32所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；或
    iv)重链可变区包含选自如SEQ ID NO：33、34和35氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：33、34和35所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：36、37和38氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：36、37和38所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体；
    或
    v)重链可变区包含选自如SEQ ID NO：39、40和41氨基酸序列所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：39、40和41所示的HCDR1、HCDR2和HCDR3分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：42、43和44氨基酸序列所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：42、43和44所示的LCDR1、LCDR2和LCDR3分别具有3，2或1个氨基酸差异的LCDR变体。
  2. 如权利要求1所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体是重组抗体，优选选自鼠源抗体、嵌合抗体、人源化抗体的重组抗体。
  3. 如权利要求2所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体轻链和重链可变区上的轻链和重链FR区序列分别来源于人种系轻链和重链或其突变序列。
  4. 如权利要求3所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:45、51、56、64或71所示的重链可变区；或其重链可变区变体，所述变体是在SEQ ID NO:45、51、56、64或71所示的重链可变区序列上具有1-10个氨基酸变化。
  5. 如权利要求4所述的单克隆抗体或其抗原结合片段，其中所述变体是在SEQ ID NO:45、51、56、64或71所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自：
    在SEQ ID NO:45所示的重链可变区上的N84S、S85R和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:51所示的重链可变区上的M48I、R72V、V79A和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:56所示的重链可变区上的Y27F、M48I、R72V、V79A、S84N和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:64所示的重链可变区上的R38K、R67K、R72V、T74K、M48I、V68A、M70L、V79A和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:71所示的重链可变区上的G27Y、M48I、L83F、A97T和其任意组合的氨基酸回复突变。
  6. 如权利要求3所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自：vi)SEQ ID NO:45或50所示的重链可变区；
    vii)SEQ ID NO:51、54至55中任一个所示的重链可变区；
    viii)SEQ ID NO:56、61、62和63中任一个所示的重链可变区；
    ix)SEQ ID NO:64、67、68、69和70中任一个所示的重链可变区；和
    x)SEQ ID NO:71、75、76和77中任一个所示的重链可变区。
  7. 如权利要求3所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:46、52、57、65或72所示的轻链可变区；或其轻链可变区变体，所述变体是在SEQ ID NO:46、52、57、65或72所示的轻链可变区上具有1-10个氨基酸变化的序列。
  8. 如权利要求7所述的单克隆抗体或其抗原结合片段，其中所述变体是在SEQ ID NO:46、52、57、65或72所示的轻链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自：
    在SEQ ID NO:46所示的轻链可变区上的S60D、T85D、A43S、S63T和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:52所示的轻链可变区上的A43S的氨基酸回复突变；
    或在SEQ ID NO:57所示的轻链可变区上的Q3V、A43S、S60D、Y87F和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:65所示的轻链可变区上的A43S、I48V和其任意组合的氨基酸回复突变；
    或在SEQ ID NO:72所示的轻链可变区上的N22S、P49S和其任意组合的氨基酸回复突变。
  9. 如权利要求3所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自：
    xi)SEQ ID NO:46、47、48和49中任一个所示的轻链可变区；
    xii)SEQ ID NO:52或53所示的轻链可变区；
    xiii)SEQ ID NO:57、58、59和60中任一个所示的轻链可变区；
    xiv)SEQ ID NO:65或66所示的轻链可变区；或
    xv)SEQ ID NO:72、73和74中任一个所示的轻链可变区。
  10. 如权利要求3所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自：
    xvi)SEQ ID NO:45或50所示的重链可变区和SEQ ID NO:46、47、48和49中任一个所示的轻链可变区；
    xvii)SEQ ID NO:51，54和55中任一个所示的重链可变区和SEQ ID NO:52或53所示的轻链可变区；
    xviii)SEQ ID NO:56、61、62和63中任一个所示的重链可变区和SEQ ID NO:57、58、59和60中任一个所示的轻链可变区；
    xix)SEQ ID NO:64、67、68、69和70中任一个所示的重链可变区和SEQ ID NO:65或66所示的轻链可变区；或
    xx)SEQ ID NO:71、75、76和77中任一个所示的重链可变区和SEQ ID NO:72、73和74中任一个所示的轻链可变区。
  11. 如权利要求1-10任一项所述的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，优选包含SEQ ID NO:78所示的人抗 体重链恒定区和SEQ ID NO:79所示的人轻链恒定区。
  12. 权利要求1至10任一项所述的单克隆抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
  13. 一种分离的单克隆抗体或其抗原结合片段，其特征在于与权利要求1-12所述的单克隆抗体或其抗原结合片段竞争结合人TIGIT。
  14. 一种药物组合物，其含有治疗有效量的根据权利要求1至13任一项所述的单克隆抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  15. 一种分离的核酸分子，其编码权利要求1至13任一项所述的单克隆抗体或其抗原结合片段。
  16. 一种重组载体，其包含权利要求15所述的核酸分子。
  17. 一种用根据权利要求16所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
  18. 用于生产权利要求1至13任一项所述的单克隆抗体或其抗原结合片段的方法，所述方法包括将权利要求17所述的宿主细胞在培养物中进行培养以形成并积累权利要求1至13任一项的单克隆抗体或其抗原结合片段，以及从培养物回收所述单克隆抗体或其抗原结合片段。
  19. 用于检测或测定人TIGIT的方法，所述方法包括使用权利要求1至13任一项所述的单克隆抗体或其抗原结合片段的步骤。
  20. 用于检测或测定人TIGIT的试剂，所述试剂包含权利要求1至13任一项所述的单克隆抗体或其抗原结合片段。
  21. 如权利要求1至13任一项所述的单克隆抗体或其抗原结合片段、如权利要求14所述的药物组合物、或如权利要求15所述的核酸分子在制备用于治疗与人TIGIT相关的疾病的药物中的用途，其中所述疾病优选是T细胞功能障碍性病症，更优选是肿瘤、癌症、免疫性疾病或感染性病症。
  22. 一种治疗与人TIGIT相关的疾病的方法，所述方法包括向受试者施用药物有效量的权利要求1至13任一项所述的单克隆抗体或其抗原结合片段，或包含权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子，以治疗人TIGIT相关的疾病，所述疾病优选是T细胞功能障碍性病症，更优选是肿瘤、癌症或感染性病症，最优选CD155阳性或PVR阳性的肿瘤、癌症或感染性病症。
  23. 一种治疗疾病的方法，所述方法包括向受试者施用药物有效量的权利要求1至13任一项所述的单克隆抗体或其抗原结合片段，或包含权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子，所述疾病优选是T细胞功能障碍性病症，更优选是肿瘤、癌症或感染性病症，最优选CD155阳性或PVR阳性的肿瘤、癌症或感染性病症。
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