WO2020108660A1 - 抗人tim-3单克隆抗体及其应用 - Google Patents

抗人tim-3单克隆抗体及其应用 Download PDF

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WO2020108660A1
WO2020108660A1 PCT/CN2019/122471 CN2019122471W WO2020108660A1 WO 2020108660 A1 WO2020108660 A1 WO 2020108660A1 CN 2019122471 W CN2019122471 W CN 2019122471W WO 2020108660 A1 WO2020108660 A1 WO 2020108660A1
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Prior art keywords
antibody
variable region
chain variable
tim
heavy chain
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PCT/CN2019/122471
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English (en)
French (fr)
Inventor
宋宁宁
段清
刘礼乐
杨达志
徐丽娜
刘虎
卫培培
王倩
王远东
邵晓慧
胡少平
张瑜
吴建
王美玲
王冬旭
戴朝晖
王梦莹
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上海开拓者生物医药有限公司
钜川生物医药
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Priority to CN201980079364.4A priority Critical patent/CN113166251B/zh
Priority to US17/298,817 priority patent/US20220033501A1/en
Priority to EP19889992.4A priority patent/EP3901171A4/en
Priority to JP2021531518A priority patent/JP2022508309A/ja
Publication of WO2020108660A1 publication Critical patent/WO2020108660A1/zh

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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • 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
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    • C07K2317/74—Inducing cell proliferation
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the field of biomedicine, in particular to a TIM-3 antibody and its preparation method and application
  • T cell immunoglobulin domain and mucin domain 3 (TIM-3, HAVCR2) is a type 301 amino acid membrane protein and is one of the known major immune checkpoints (Immune Checkpoint).
  • TIM-3 is mainly expressed in activated CD4 + and CD8 + T lymphocytes, natural regulatory T cells, NK cells, and innate cells (macrophages, monocytes, and dendritic cells).
  • TIM-3 ligands include phosphatidylserine (PtdSer), galectin-9 (Gal-9), high mobility group box 1 (HMGB1) and carcinoembryonic antigen cell adhesion molecule 1 (CECAM1 ).
  • TIM-3 plays a role in regulating all aspects of the immune response by binding to its ligand.
  • the interaction of TIM-3 and CECAM1 inhibits T cell activation and promotes T cell tolerance and depletion.
  • Regulatory T cells and many tumor cells express Gal-9 protein and bind to TIM-3 to inhibit the activation of effector T cells.
  • the combination of HMGB1 and TIM-3 acts on antigen-presenting cells and innate cells to reduce the inflammatory response mediated by injury-related molecular patterns.
  • TIM-3 binds phosphatidylserine to promote the elimination of apoptotic cells.
  • TIM-3 abnormal expression of TIM-3 is closely related to many diseases. Studies have found that in patients with HIV, HCV infection and some tumor patients, the expression of TIM-3 in T cells is increased, which mediates the apoptosis of T effector cells and transmits negative regulatory signals, which leads to disorders of the immune system and paralysis of the body's immune function. In addition, the tumor-infiltrating lymphocytes in various mouse tumor models co-express TIM-3 and PD-1, and exhibit functional exhaustion, loss of proliferation, and secretion of some cytokines (IL-2, TNF- ⁇ , and IFN - ⁇ ) ability.
  • IL-2 cytokines
  • blocking PD-1 and TIM-3 signaling pathways alone can more effectively inhibit tumor growth than blocking PD-1 or TIM-3 signaling pathways alone.
  • TIM-3 - / PD-1 - the CD8 + T Cell functions are more disordered and the ability to secrete cytokines is weaker.
  • TIM-3 antibodies at home and abroad are in clinical research stage, and other antibody drugs are still in the stage of discovery and research.
  • There is no TIM-3 antibody in clinical application and it is urgent to develop a TIM with good activity, wide indications and high yield.
  • -3 antibody It is urgent to develop a more active TIM-3 antibody to further improve the therapeutic effect.
  • the activity of the antibody itself is affected by the variable region sequence and the structure of the constant region.
  • the variable region sequence of an antibody determines the determinants of antigen recognition, binding affinity and metabolic rate in the body, which will affect its in vivo activity and even the clinical effect of different patients.
  • TIM-3 antibody is mainly used for the treatment of malignant solid tumors and lymphomas, and it is also mainly concentrated on its combined use with other therapies or target drugs to develop antibodies with wide indications to expand its clinical application. Symptoms, it is urgent to develop TIM-3 antibodies with higher output to reduce the cost of treatment for patients and benefit more patients. In addition, tumor immunotherapy is currently expensive, and there is an urgent need to invent and produce new antibodies to reduce costs.
  • the present invention provides a TIM-3 antibody with high affinity and strong specificity, and a preparation method and use thereof.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4, and
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • n is independently 0, 1, 2, 3, 4, 5, or 6;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid and can retain the binding affinity of TIM-3.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 10n+1, where n is 0, 1, 2, 3, 4, 5, or 6.
  • a heavy chain of an antibody having a heavy chain variable region as described in the first aspect of the present invention.
  • the heavy chain further includes a heavy chain constant region.
  • the heavy chain constant region is of human origin.
  • the heavy chain constant region is a human antibody heavy chain IgG4 constant region.
  • a light chain variable region of an antibody comprising the following three complementarity determining regions CDR:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • n is independently 0, 1, 2, 3, 4, 5, or 6;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO. 10n+6, where n is 0, 1, 2, 3, 4, 5, or 6.
  • a light chain of an antibody having a light chain variable region as described in the third aspect of the present invention.
  • the light chain further includes a light chain constant region.
  • the light chain constant region is human.
  • the light chain constant region is a human antibody light chain kappa constant region.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention,
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • the amino acid sequence of any of the above CDRs includes derived, deleted, modified, and/or substituted 1, 2 or 3 amino acid derived CDR sequences, and the VH and VL containing the derived CDR sequences are included
  • the derived antibody can retain the affinity of binding to TIM-3.
  • the ratio (F1/F0) of the affinity F1 of the derived antibody to TIM-3 to the affinity of the corresponding non-derivatized antibody to TIM-3 is 0.5-2, preferably 0.7-1.5, and better still 0.8-1.2.
  • the number of added, deleted, modified and/or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).
  • the derivative sequence added, deleted, modified, and/or substituted for at least one amino acid and capable of retaining the binding affinity of TIM-3 is an amino acid with homology or sequence identity of at least 96% sequence.
  • the antibody further includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.
  • the heavy chain constant region is a human antibody heavy chain IgG4 constant region
  • the light chain constant region is a human antibody light chain kappa constant region
  • the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or a combination thereof.
  • the ratio of the immunogenicity Z1 of the chimeric antibody in humans to the immunogenicity Z0 of non-chimeric antibodies (such as murine antibodies) in humans is 0 -0.5, preferably 0-0.2, more preferably 0-0.05 (e.g. 0.001-0.05).
  • the antibody is a partially or fully humanized, or fully human monoclonal antibody.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is an antibody full-length protein or an antigen-binding fragment.
  • the antibody is a bispecific antibody or a multispecific antibody.
  • the antibody has one or more characteristics selected from the group consisting of:
  • the antibody has a heavy chain variable region according to the first aspect of the present invention and a light chain variable region according to the third aspect of the present invention;
  • heavy chain variable region and the light chain variable region include CDRs selected from the group consisting of:
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • the antibody has a heavy chain variable region according to the first aspect of the present invention and a light chain variable region according to the third aspect of the present invention; wherein,
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO.3,
  • VH-CDR2 shown in SEQ ID NO. 4, and
  • VH-CDR3 shown in SEQ ID NO.5;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO. 9, and
  • VL-CDR3 shown in SEQ ID NO.10;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.14, and
  • VH-CDR3 shown in SEQ ID NO.15;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.19, and
  • VL-CDR3 shown in SEQ ID NO.20;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.24, and
  • VH-CDR3 shown in SEQ ID NO.25;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.29.
  • VL-CDR3 shown in SEQ ID NO.30;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO. 34, and
  • VH-CDR3 shown in SEQ ID NO.35;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO. 39.
  • VL-CDR3 shown in SEQ ID NO.40;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO. 44, and
  • VH-CDR3 shown in SEQ ID NO.45;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.49.
  • VL-CDR3 shown in SEQ ID NO.50;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.54, and
  • VH-CDR3 shown in SEQ ID NO.55;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO. 59.
  • VL-CDR3 shown in SEQ ID NO.60;
  • the heavy chain variable region includes the following three complementarity determining regions CDR:
  • VH-CDR2 shown in SEQ ID NO.64, and
  • VH-CDR3 shown in SEQ ID NO.65;
  • the light chain variable region includes the following three complementarity determining regions CDR:
  • VL-CDR2 shown in SEQ ID NO.69.
  • VL-CDR3 shown in SEQ ID NO.70.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 1, 11, 21, 31, 41, 51, or 61; and/or the light chain of the antibody
  • the variable region contains the amino acid sequence shown in SEQ ID NO. 6, 16, 26, 36, 46, 56 or 66.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.1; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.6.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.11; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.16.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 21; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 26.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 31; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 36.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 41; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 46.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 51; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 56.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 61; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 66.
  • the antibody is selected from the group consisting of:
  • amino acid sequence of the heavy chain variable region and the amino acid sequence shown in SEQ ID NO.1, 11, 21, 31, 41, 51, or 61 in the sequence listing are at least 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • amino acid sequence of the light chain variable region and the amino acid sequence shown in SEQ ID NO. 6, 16, 26, 36, 46, 56 or 66 in the sequence listing are at least 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • a recombinant protein in a sixth aspect of the present invention, includes:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, dimer, or polymer.
  • the recombinant protein includes:
  • polynucleotide encoding the heavy chain variable region is shown in SEQ ID NO. 2, 12, 22, 32, 42, 52, or 62; and/or, the light chain may be encoded
  • the polynucleotide of the variable region is shown in SEQ ID NO. 7, 17, 27, 37, 47, 57 or 67.
  • polynucleotide encoding the heavy chain variable region sequence and the polynucleotide encoding the light chain variable region sequence are selected from the group consisting of:
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell containing the vector or genome of the eighth aspect of the present invention incorporating the polynucleoside of the seventh aspect of the present invention acid.
  • an antibody conjugate comprising:
  • an antibody portion selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, and a third according to the invention
  • the light chain variable region of the aspect, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention, or a combination thereof and (b) partially coupled with the antibody
  • the coupling part is selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
  • the antibody part and the coupling part are coupled by a chemical bond or a linker.
  • an immune cell which expresses or is exposed to the cell membrane the antibody of the fifth aspect of the present invention.
  • the immune cells include NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, and the third according to the invention
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition includes 0.01-99.99% of the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, and the tenth according to the invention
  • the antibody conjugate, the immune cell according to the eleventh aspect of the present invention, or a combination thereof and a pharmaceutical carrier of 0.01 to 99.99% the percentage is a mass percentage of the pharmaceutical composition.
  • a use of an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present invention, and a second aspect of the present invention The heavy chain, the light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, such as the present
  • preparation of diagnostic reagents or kits and/or (b) preparation of drugs for preventing and/or treating diseases related to abnormal expression or function of TIM-3.
  • the diagnostic reagent is a test piece or a test plate.
  • the diseases related to abnormal expression or function of TIM-3 are selected from the group consisting of tumors and autoimmune diseases.
  • the tumor is selected from the group consisting of melanoma, mesothelioma, non-small cell lung cancer, breast cancer, liver cancer, synovial sarcoma, metastatic colon cancer, kidney cancer, bladder cancer, prostate cancer, Ovarian cancer, chronic hepatitis C virus infection, advanced solid cancer, malignant tumor of digestive organs, endometrial cancer, recurrent melanoma, squamous cell carcinoma of the head and neck, cutaneous T-cell lymphoma, fallopian tube cancer, peritoneal tumor, muscle infiltration Bladder cancer, extensive stage small cell lung cancer, adult acute myeloid leukemia, atypical chronic myeloid leukemia, ovarian epithelial cell carcinoma, B-cell chronic lymphocytic leukemia, cutaneous B-cell non-Hodgkin's lymphoma, intraocular lymphoma Tumors, testicular choriocarcinoma, neuroblastoma, esophageal cancer
  • the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, xerophthalmia syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, polyarteritis nodosa , Wegener granulomatosis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, pemphigus vulgaris, pemphigoid, transplant rejection.
  • the diagnostic reagent or kit is used to detect the TIM-3 protein in the sample.
  • the diagnostic reagent or kit is used to diagnose TIM-3 related diseases.
  • the diagnostic reagent or kit is used to detect the TIM-3 protein in the sample.
  • a fourteenth aspect of the present invention there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of TIM-3 protein in a sample, the method comprising the steps of:
  • composition for in vitro detection of TIM-3 protein in a sample which comprises the antibody according to the fifth aspect of the present invention, and the recombinant according to the sixth aspect of the present invention
  • the protein, the antibody conjugate according to the tenth aspect of the present invention, the immune cell according to the eleventh aspect of the present invention, or a combination thereof are used as active ingredients.
  • a detection board comprising: a substrate (supporting plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the invention 2.
  • kit includes:
  • a first container containing the antibody of the present invention and/or
  • the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide comprising:
  • the recombinant polypeptide is isolated from the culture, and the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.
  • a pharmaceutical combination including:
  • a first active ingredient comprising the antibody 1 according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the tenth aspect according to the invention
  • a second active ingredient which includes a second antibody or a chemotherapeutic agent.
  • the second antibody is selected from the group consisting of CTLA4 antibody and PD-1 antibody.
  • the second antibody is PD-1 antibody.
  • the chemotherapeutic agent is selected from the group consisting of docetaxel, carboplatin, or a combination thereof.
  • the antibody according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention, or the antibody conjugate according to the tenth aspect of the invention Or the combination of the immune cell according to the eleventh aspect of the present invention, and/or the pharmaceutical composition according to the twelfth aspect of the present invention and a second antibody or chemotherapeutic agent is prepared for the treatment of TIM-3 expression or function Use of drugs in abnormally related diseases.
  • the second antibody is selected from the group consisting of CTLA4 antibody and PD-1 antibody.
  • the second antibody is PD-1 antibody.
  • a method for treating a disease associated with abnormal expression or function of TIM-3 an effective amount of the antibody according to the fifth aspect of the present invention is administered to a subject in need, Or the recombinant protein according to the sixth aspect of the present invention, or the antibody conjugate according to the tenth aspect of the present invention, or the immune cell according to the eleventh aspect of the present invention, or according to the present invention
  • the pharmaceutical composition according to the twelfth aspect, or a combination thereof is administered to a subject in need, Or the recombinant protein according to the sixth aspect of the present invention, or the antibody conjugate according to the tenth aspect of the present invention, or the immune cell according to the eleventh aspect of the present invention, or according to the present invention.
  • the disease associated with abnormal expression or function of TIM-3 is cancer.
  • the method further includes: administering a safe and effective amount of the second antibody to the subject before, during and/or after administration of the first active ingredient.
  • the second antibody is selected from the group consisting of PD-1 antibody and CTLA4 antibody.
  • the second antibody is PD-1 antibody.
  • Figure 1 shows the binding activity of TIM-3-hFc protein and its commercial antibody.
  • Figure 2 shows the results of FACS detection of HEK293 cells transfected with TIM-3 gene.
  • Fig. 3 shows the serum antibody titer of TIM-3-hFC protein immunized by ELISA.
  • Fig. 4 shows the reactivity of TIM-3 antibody with human TIM-3 extracellular domain protein in an enzyme-linked immunosorbent assay.
  • Figure 5 shows the reactivity of TIM-3 antibody with protein in the extracellular domain of monkey TIM-3 in an enzyme-linked immunosorbent assay.
  • Figure 6 shows the reactivity of the TIM-3 antibody with the protein in the extracellular domain of murine TIM-3 in an enzyme-linked immunosorbent assay.
  • Figure 7 shows FACS detection of the binding reaction of TIM-3 antibody to CHOK1-hTIM-3.
  • Figure 8 shows the FACS detection of the binding reaction of TIM-3 antibody to CHOK1-cTIM-3.
  • Figure 9 shows the effect of antibodies on IFN- ⁇ secretion in the OKT3-dependent PBMC activation test.
  • Figure 10 shows the reactivity of fully human TIM-3 antibody with human TIM-3 extracellular domain protein in an enzyme-linked immunosorbent assay.
  • Figure 11 shows the reactivity of fully human TIM-3 antibody with protein in the extracellular domain of monkey TIM-3 in an enzyme-linked immunosorbent assay.
  • Figure 12 shows the reactivity of fully human TIM-3 antibody with mouse TIM-3 extracellular domain protein in an enzyme-linked immunosorbent assay.
  • Figure 13 shows FACS detection of the binding reaction of fully human TIM-3 antibody to CHOK1-hTIM-3.
  • Figure 14 shows the FACS detection of the binding reaction of fully human TIM-3 antibody to CHOK1-cTIM-3.
  • Figure 15 shows that fully human TIM-3 antibody blocks the binding of TIM-3 protein to its receptor phosphatidylserine.
  • Figure 16 shows the effect of fully human TIM-3 antibody on IFN- ⁇ secretion in a lymphocyte activation test (donor X).
  • Figure 17 shows the effect of fully human TIM-3 antibody on IFN- ⁇ secretion in a lymphocyte activation test (donor Y).
  • the present inventors used human-derived TIM-3 as an immunogen and used optimized hybridoma technology to prepare TIM-3 antibody.
  • the present invention uses human antibody transgenic mouse technology for the preparation of fully human antibodies to obtain lead antibodies for TIM-3 antibody; and through preliminary production, purification and verification of lead antibodies, high antibody affinity, TIM-3 antibody with excellent biological characteristics such as significant increase in IFN expression level in human peripheral blood mononuclear cell activation reaction; then sequenced by molecular biology method to know the heavy chain variable region and light chain variable of TIM-3 antibody The amino acid sequence of the region.
  • the TIM-3 antibody has high affinity with human and monkey-derived TIM-3 proteins, and can increase the expression level of IFN in human peripheral blood mononuclear cells induced by OKT3 activation.
  • the present invention also provides the use of these antibodies, including but not limited to inhibiting the negative regulation of TIM-3 and its ligand-mediated signaling pathways, activating tumor-specific immune responses, alone or in combination with anti-PD-1, CTLA-4 monoclonal antibodies Or other anti-tumor drugs are used in combination for tumor immunotherapy. On this basis, the present invention has been completed.
  • VH-CDR1 and “CDR-H1” are used interchangeably, both refer to the CDR1 of the heavy chain variable region;
  • VH-CDR2 and “CDR-H2” are used interchangeably, both refer to the heavy chain CDR2 of the variable region;
  • VH-CDR3 and “CDR-H3” are used interchangeably, and both refer to the CDR3 of the variable region of the heavy chain.
  • VL-CDR1 and CDR-L1 can be used interchangeably, both refer to CDR1 of the light chain variable region;
  • VL-CDR2 and CDR-L2 can be used interchangeably, both refer to the light chain variable region CDR2;
  • VL-CDR3 and CDR-L3 are used interchangeably and both refer to CDR3 of the light chain variable region.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 Daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced disulfide bonds in the chain. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
  • variable means that certain parts of the variable region of an antibody differ in sequence, which forms the binding and specificity of various specific antibodies for their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called the complementarity determining region (CDR) or hypervariable region in the light chain and heavy chain variable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining region
  • FR framework region
  • the variable regions of the natural heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of the ⁇ -sheet structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669) (1991)).
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
  • immunoglobulins The "light chain" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of its constant region. According to the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be divided into different types. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • immunoglobulins There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which divide this segment into 4 framework regions (FR)
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in space structure, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention includes not only whole antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombination techniques, and they are all useful antibodies.
  • a chimeric antibody is a molecule in which different parts come from different animal species, such as a variable region with a monoclonal antibody from a mouse, and a chimeric antibody from a constant region of a human immunoglobulin (see, for example, U.S. Patent No. US Patent 4,816,397, hereby incorporated by reference in its entirety).
  • Humanized antibodies refer to antibody molecules derived from non-human species, having one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see US Patent 5,585,089, It is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • CDRs complementarity determining regions
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes conservative variants, which means that there are at most 10, preferably at most 8, more preferably at most 5, compared with the amino acid sequence of the antibody of the present invention, and most preferably Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitution according to Table 19.
  • the antibody is an anti-TIM-3 antibody.
  • the present invention provides a high specificity and high affinity antibody against TIM-3, which includes a heavy chain and a light chain, the heavy chain contains a heavy chain variable region (VH) amino acid sequence, and the light chain contains a light chain Variable region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the heavy chain variable region (VH) has a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4, and
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • n is independently 0, 1, 2, 3, 4, 5, or 6;
  • the light chain variable region (VL) has a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • n is independently 0, 1, 2, 3, 4, 5, or 6;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • the heavy chain variable region (VH) includes the following three complementarity determining regions CDR:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4, and
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • the light chain variable region (VL) includes the following three complementarity determining regions CDR:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • n is independently 0, 1, 2, 3, 4, 5 or 6; preferably n is 0 or 1;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • sequence formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably The amino acid sequence is at least 90%, most preferably at least 95%.
  • the preferred method of determining identity is to obtain the greatest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to: GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990).
  • the BLASTX program is available to the public from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • the antibody in the present invention may be a full-length protein (such as IgG1, IgG2a, IgG2b or IgG2c), or may be a protein fragment containing an antigen-antibody binding domain (such as Fab, F(ab'), sdAb, ScFv fragment).
  • the antibody in the present invention may be a wild-type protein or a mutant protein that has achieved a specific effect through a specific mutation, for example, the mutation is used to eliminate the effector function of the antibody.
  • the antibodies described herein are full-length antibody antibodies, antigen-antibody binding domain protein fragments, bispecific antibodies, multispecific antibodies, single chain antibodies (single chain antibody, scFv), single domain antibodies (single domain antibody) , SdAb) and single-region antibodies (Signle-domain antibodies) one or more, and monoclonal antibodies or polyclonal antibodies prepared by the above antibodies.
  • the monoclonal antibody can be developed in various ways and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody full-length protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a full human antibody full-length protein.
  • the full-length antibody is IgG1, IgG2, IgG3 or IgG4.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, more preferably fully human Source antibody.
  • the antibody derivative of the present invention may be a single chain antibody, and/or antibody fragments, such as: Fab, Fab', (Fab') 2 or other known antibody derivatives in the art, and IgA, IgD, IgE , IgG and IgM antibodies or other subtypes of any one or several.
  • the single chain antibody is a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the animal is preferably a mammal, such as a rat.
  • the antibodies of the present invention may be chimeric antibodies, humanized antibodies, CDR grafted and/or modified antibodies targeting TIM-3 (eg, human TIM-3).
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the original amino acid sequence, more preferably not more than 35%, more preferably 1-33% , More preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of the added, deleted, modified and/or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably For 1-2.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 1, 11, 21, 31, 41, 51, or 61.
  • the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO. 6, 16, 26, 36, 46, 56 or 66.
  • the amino acid sequence of the heavy chain variable region (VH) of the antibody targeting TIM-3, and/or the light chain variable region amino acid sequence is shown in Table 20 below:
  • the antibody targeting TIM-3 is 7A4F10, 10D2H2, 134H3G6, 215A8F2, 34B6D8, 39E5H1, 57F4E5.
  • the antibody targeting TIM-3 is 7A4F10.
  • the antibody targeting TIM-3 is 10D2H2.
  • the present invention also provides a recombinant protein comprising one or more of heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of TIM-3 antibody, And/or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of the TIM-3 antibody,
  • VH-CDR1 VH-CDR1
  • VH-CDR2 heavy chain CDR2
  • VH-CDR3 VH-CDR3
  • the sequence of the heavy chain CDR1-3 is as follows:
  • VH-CDR1 shown in SEQ ID NO.10n+3,
  • VH-CDR2 shown in SEQ ID NO.10n+4,
  • VH-CDR3 shown in SEQ ID NO.10n+5;
  • the sequence of the light chain CDR1-3 is as follows:
  • VL-CDR1 shown in SEQ ID NO.10n+8,
  • VL-CDR2 shown in SEQ ID NO.10n+9
  • VL-CDR3 shown in SEQ ID NO.10n+10;
  • n is independently 0, 1, 2, 3, 4, 5 or 6; preferably n is 0 or 1;
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • sequence formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably The amino acid sequence is at least 90%, most preferably at least 95%.
  • the recombinant protein of the present invention includes the heavy chain variable region of the TIM-3 antibody and/or the light chain variable region of the TIM-3 antibody, and the heavy chain variable region of the antibody contains SEQ ID No. 1, 11, 21, 31, 41, 51 or 61; the light chain variable region of the antibody contains SEQ ID No. 6, 16, 26, 36, 46, 56 or 66 The amino acid sequence shown.
  • the recombinant protein of the present invention includes the heavy chain variable region of the TIM-3 antibody and the light chain variable region of the TIM-3 antibody.
  • the heavy chain variable region of the antibody contains SEQ ID NO .1, 11, 21, 31, 41, 51 or 61
  • the light chain variable region of the antibody contains SEQ ID NO. 6, 16, 26, 36, 46, 56 or 66 Amino acid sequence.
  • sequence numbers of the amino acid sequences of the recombinant protein and the heavy chain CDR1-3 and light chain CDR1-3 included in the recombinant protein are shown in Table 21:
  • any one of the above-mentioned amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and/or substituted for at least one amino acid, and can retain the binding affinity of TIM-3.
  • the recombinant protein further includes an antibody heavy chain constant region and/or an antibody light chain constant region.
  • the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region Or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, and more preferably a human antibody light chain constant region.
  • the recombinant protein is a conventional protein in the art, preferably, it is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody, fragment, scFv) ), single domain antibody (single domain antibody, sdAb) and single domain antibody (Signle-domain antibody) one or more, and monoclonal antibody or polyclonal antibody prepared by the above antibody.
  • the monoclonal antibody can be developed in various ways and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody full-length protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a full human antibody full-length protein.
  • the full-length antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single chain antibody is a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragments are conventional antigen-antibody binding domain protein fragments in the art, which include the light chain variable region, the light chain constant region, and the Fd segment of the heavy chain constant region.
  • the protein fragments of the antigen-antibody binding domain are Fab and F(ab').
  • the single domain antibody is a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
  • the single-region antibody is a conventional single-region antibody in the art, which includes only the heavy chain variable region.
  • the preparation method of the recombinant protein is a conventional preparation method in the art.
  • the preparation method is preferably obtained by separation from an expression transformant that recombinantly expresses the protein or by artificially synthesizing the protein sequence.
  • the method for separating and obtaining the recombinant transformant that expresses the protein is preferably as follows: clone the nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, and transform the resulting recombinant vector into a transformant to obtain recombinant expression
  • the transformant can be isolated and purified to obtain the recombinant protein by culturing the obtained recombinant expression transformant.
  • the present invention also provides a nucleic acid encoding the heavy chain variable region or light chain variable region of the above-mentioned antibody (eg, anti-TIM-3 antibody) or recombinant protein or anti-TIM-3 antibody.
  • a nucleic acid encoding the heavy chain variable region or light chain variable region of the above-mentioned antibody (eg, anti-TIM-3 antibody) or recombinant protein or anti-TIM-3 antibody.
  • nucleotide sequence of the nucleic acid encoding the variable region of the heavy chain is shown in SEQ ID NO. 2, 12, 22, 32, 42, 52 or 62 of the Sequence Listing; and/or The nucleotide sequence of the nucleic acid of the chain variable region is shown in SEQ ID NO. 7, 17, 27, 37, 47, 57 or 67 in the sequence listing.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO. 2, 12, 22, 32, 42, 52 or 62 of the Sequence Listing; and the light chain variable is encoded
  • the nucleotide sequence of the nucleic acid in the region is shown in SEQ ID NO. 7, 17, 27, 37, 47, 57 or 67 of the Sequence Listing.
  • the preparation method of the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis .
  • the base sequence encoding the amino acid sequence of the above-mentioned protein may be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog.
  • the homologue of the polynucleotide in the present invention can be prepared by replacing, deleting, or adding one or more bases of the gene encoding the protein sequence within the range of maintaining antibody activity.
  • the invention also provides a recombinant expression vector containing the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, it is constructed by connecting the nucleic acid molecule of the present invention to various expression vectors.
  • the expression vector is a variety of conventional vectors in the art, as long as it can contain the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: it is prepared by transforming the above-mentioned recombinant expression vector into a host cell.
  • the host cell is a variety of conventional host cells in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleic acid carried can be effectively expressed.
  • the host cell is E. coli TG1 or E. coli BL21 cells (expressing single chain antibody or Fab antibody), or HEK293 or CHO cells (expressing full-length IgG antibody).
  • the conversion method is a conventional conversion method in the art, preferably a chemical conversion method, a heat shock method or an electric conversion method.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single chain antibody.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.
  • synthetic methods can be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the DNA sequence encoding the antibody (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the invention by chemical synthesis.
  • the present invention also relates to vectors containing the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
  • the transformed host cells are cultured under conditions suitable for expression of the antibody of the present invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, etc.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to the personnel.
  • the obtained monoclonal antibody can be identified by conventional means.
  • the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding tests (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).
  • the binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibody of the present invention can be expressed in a cell, on a cell membrane, or secreted out of a cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with protein precipitation agent (salting out method)
  • centrifugation osmotic disruption
  • ultrasonic treatment ultracentrifugation
  • molecular sieve chromatography gel filtration
  • adsorption layer Analysis ion exchange chromatography
  • ADC Antibody-drug conjugate
  • the invention also provides antibody-drug conjugate (ADC) based on the antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-coupled drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a drug having therapeutic activity.
  • the effector molecule may be one or more of toxic protein, chemotherapeutic drug, small molecule drug or radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent using a carboxyl group may be any one or several of cis-aconitic anhydride type coupling agents (such as cis-aconitic anhydride) and acylhydrazone type coupling agents (coupling sites are acylhydrazones).
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg drugs, detection reagents, stabilizers
  • the functional agent may be directly or indirectly linked to the antibody via a linker.
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and antibody.
  • the joint may be degradable or non-degradable.
  • the degradable linker is typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degraded linkers, including peptide group-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide Enzymatically degraded glucuronide-containing linker.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that can degrade under reducing conditions (such as disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by a protease.
  • the linker Before connecting to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Mercapto-specific reactive reactive groups are preferred and include: for example maleimides, halogenated amides (eg iodine, bromine or chlorinated); halogenated esters (eg iodine, bromine or chlorinated) ); halogenated methyl ketone (such as iodine, bromine or chloro), benzyl halide (such as iodine, bromine or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3,6- Di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker may include, for example, maleimide attached to the antibody through thi
  • the drug may be any drug that is cytotoxic, inhibits cell growth, or immunosuppresses.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug may have an amino group, a carboxyl group, a mercapto group, a hydroxyl group, or a ketone group that can form a bond with the linker.
  • the drug is directly connected to the linker, the drug has a reactive active group before being connected to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylation reagents, and tubulin inhibitors
  • typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes
  • benzodiazepines or benzodiazepine containing drugs such as pyrrolo[1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines) and vinca alkaloids (vinca alkaloids).
  • PBDs pyrrolo[1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • vinca alkaloids vinca alkaloids
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two-step or multi-step method.
  • the cysteine residue reacts with the reactive part of the linker in the first step, and in the subsequent step, the functional group on the linker reacts with the drug, thereby forming an ADC.
  • the functional group on the linker is selected to facilitate specific reaction with the appropriate reactive group on the drug moiety.
  • the azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety.
  • the drug is covalently bonded to the linker through 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • React with alcohols and activated esters, such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • activated esters such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody and the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention include: binding the antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the method of the present invention further includes: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • the present invention also provides the use of the antibody, antibody conjugate ADC, recombinant protein, and/or immune cell of the present invention, for example, for preparing a diagnostic preparation or preparing a medicine.
  • the medicine is a medicine for preventing and/or treating diseases related to abnormal expression or function of TIM-3.
  • antibodies, ADCs, recombinant proteins, and/or immune cells of the present invention include (but are not limited to):
  • the tumors include (but are not limited to): melanoma, mesothelioma, non-small cell lung cancer, breast cancer, liver cancer, synovial sarcoma, metastatic colon cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, C Hepatitis virus chronic infection, advanced solid cancer, malignant tumor of digestive organs, endometrial cancer, recurrent melanoma, squamous cell carcinoma of the head and neck, cutaneous T-cell lymphoma, fallopian tube cancer, peritoneal tumor, muscle-invasive bladder cancer, Extensive stage small cell lung cancer, adult acute myeloid leukemia, atypical chronic myeloid leukemia, ovarian epithelial cell carcinoma, B-cell chronic lymphocytic leukemia, skin B-cell non-Hodgkin's lymphoma, intrao
  • autoimmune diseases including (but not limited to): systemic lupus erythematosus, xerophthalmia syndrome, rheumatoid arthritis, ankylosing spondylitis , Scleroderma, polyarteritis nodosa, Wegener's granulomatosis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, pemphigus vulgaris, pemphigoid, transplant rejection.
  • systemic lupus erythematosus including xerophthalmia syndrome, rheumatoid arthritis, ankylosing spondylitis , Scleroderma, polyarteritis nodosa, Wegener's granulomatosis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, pemphigus vulgaris, pemphigoid, transplant rejection.
  • the antibody of the present invention or its ADC can be used in a detection application, for example, to detect a sample, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention shall include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or an puncture or needle biopsy of an organ.
  • the samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the antibody of the present invention may be fixed to the detection plate.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, which may be aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules , Injections or infusions.
  • the antibody of the present invention may also be used for cell therapy by expressing a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and/or treating diseases related to abnormal expression or function of TIM-3.
  • the pharmaceutical composition of the present invention can be directly used to bind TIM-3 protein molecules, and thus can be used to prevent and treat tumors and other diseases.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) and pharmaceutical Acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptide of the present invention can be used together with other therapeutic agents.
  • the pharmaceutical composition of the present invention further includes one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier may be any suitable physiological or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the aforementioned protein and 0.01-99.99% of a pharmaceutical carrier, and the percentage is a mass percentage of the pharmaceutical composition.
  • the administration amount of the pharmaceutical composition is an effective amount
  • the effective amount is an amount that can alleviate or delay the progression of a disease, degenerative or traumatic condition.
  • the effective amount can be determined on an individual basis and will be based in part on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors such as individual basis and using no more than routine experimentation.
  • a safe and effective amount of an immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of skilled physicians.
  • the present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for preventing and/or treating diseases related to abnormal expression or function of TIM-3.
  • the diseases related to abnormal expression or function of TIM-3 are cancer and autoimmune diseases.
  • the present invention also provides a method for detecting TIM-3 protein in a sample (for example, detecting over-expressing TIM-3 cells), which includes the steps of contacting the above-mentioned antibody with the sample to be tested in vitro, and detecting the above-mentioned antibody with the sample It is only necessary to check whether the sample binds to form an antigen-antibody complex.
  • overexpression is conventional in the art, and refers to the overexpression of RNA or protein of the TIM-3 protein in the test sample (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation changes), and Local overexpression and increased functional activity due to changes in protein delivery patterns (increased nuclear localization) (eg, in the case of increased enzymatic hydrolysis of the substrate).
  • the detection method of whether the above forms an antigen-antibody complex is a conventional detection method in the art, preferably a flow cytometry (FACS) test.
  • FACS flow cytometry
  • the invention provides a composition for detecting TIM-3 protein in a sample, which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient.
  • a composition for detecting TIM-3 protein in a sample which comprises the above-mentioned antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient.
  • it also includes a compound composed of the functional fragments of the above-mentioned antibody as an active ingredient.
  • the present invention uses rat-human chimeric antibody transgenic mice to obtain fully human antibodies.
  • the obtained antibodies have a series of excellent characteristics:
  • variable region sequence is different from the existing antibody (homology ⁇ 92%)
  • the transgenic mice used in the present invention can obtain fully human antibodies more easily than wild-type mice, thereby reducing the immunogenicity of the antibodies; compared with fully human antibody transgenic mice, the number of antibodies obtained Strong affinity, good sequence diversity and high activity;
  • the present invention uses hybridoma technology to obtain antibodies. Compared with antibodies obtained from phage libraries, the antibodies have high affinity and good sequence expression;
  • the present invention obtains antibodies with different sequences, which can specifically bind to TIM-3, and the binding activity is lower than nanomolar. By reversing the inhibition of T cell activation activity by TIM-3, T cells are activated to secrete IFN.
  • the room temperature described in the examples is conventional room temperature in the art, and is generally 10-30°C.
  • Immunogen 1 the human TIM-3 protein extracellular region amino acid sequence 22-200 (as shown in the sequence listing SEQ ID NO.71) was cloned into a pCpC vector (purchased from Invitrogen, with human IgG Fc fragment (hFc) V044-50) and prepare plasmids according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • HEK293 cells purchased from Invitrogen
  • PI transiently transfected
  • FreeStyle TM 293 Invitrogen
  • the culture supernatant was loaded onto a protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), while monitoring the change in ultraviolet absorption value (A280nm) with an ultraviolet (UV) detector.
  • the protein affinity chromatography column was washed with PBS phosphate buffer (pH 7.2) until the UV absorption value returned to the baseline, and then eluted with 0.1 M glycine hydrochloric acid (pH 2.5) to collect the protein A affinity layer.
  • the hFc-tagged TIM-3 protein (TIM-3-hFc) eluted from the column was dialyzed against PBS phosphate buffer (pH 7.2) in a refrigerator at 4°C overnight. After dialysis, the protein was sterile filtered at 0.22 micron and then stored at -80°C to obtain purified immunogenic human TIM-3-hFc protein.
  • the immunogen TIM-3-hFc protein needs to undergo a series of quality control tests before use, such as detection of protein concentration, purity, molecular weight and biological activity.
  • TIM-3-hFc ie, immunogen
  • PBS phosphate buffer pH7.4
  • ELISA blocking solution containing 1% BSA, PBS phosphate buffer pH7.4, the percentage is the mass percentage
  • the commercial anti-TIM-3 antibody was purchased from R&D system, trade number MAB2365.
  • HRP horseradish peroxidase labeled secondary antibody
  • 100 ⁇ l/well TMB coloring solution was added.
  • 50 ⁇ l of 1N hydrochloric acid was added to stop the color reaction, and the OD450nm reading was read with an ELISA plate reader. After each step, the plates need to be washed.
  • the negative control antibody is rat IgG, the results are shown in Figure 1 and Table 1.
  • TIM-3 full-length amino acid sequence cDNA was cloned into pCDNA3.1 vector and coated onto 1.0um gold colloid bullets, and immunized with Helios gene gun (Bio-rad). The detailed method was developed according to the Helios gene gun instruction manual.
  • transgenic mice introduced human immunoglobulin variable region genes and rat immunoglobulin constant region genes, while the Ig expression of the mice themselves was silenced (F.G. Franklin, etal, patent#WO2010/070263Al). After being immunized with antigens, the transgenic mice can produce immune responses and antibody titers comparable to normal mice (such as Balb/c).
  • Immunogen 1 Immunization adopts 6-8 weeks old Harbor human antibody transgenic mice (purchased from Beijing Viton Lihua Company), and the mice are raised under SPF conditions.
  • the immunogen TIM-3-hFc protein was emulsified with Freund's complete adjuvant and injected intraperitoneally 0.25 ml, that is, each mouse was injected with 100 micrograms of immunogen protein.
  • the immunogen protein was emulsified with Freund's incomplete adjuvant and injected intraperitoneally 0.25 ml, that is, each mouse was injected with 50 micrograms of immunogen protein.
  • the interval between the first immunization and the first booster immunization is 2 weeks, and the interval between each subsequent booster immunization is 3 weeks.
  • Table 3 shows that the immunized sera of mice immunized with TIM-3-hFc all bind to the immunogen to varying degrees, showing antigen-antibody reactions, with the highest dilution of about 10,000.
  • the blank control is 1% (w/w) BSA, and the batch refers to the mouse serum on the seventh day after the second booster immunization.
  • the data in the table is the OD 450nm value.
  • the cells were counted, and the cells were diluted with phosphate buffer (pH 7.2) to 2 ⁇ 10 7 cells per ml.
  • phosphate buffer pH 7.2
  • Each mouse was injected intraperitoneally with 0.5 ml of cell suspension at each immunization.
  • the interval between the first and second immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks.
  • blood was collected one week after each immunization, and the antibody titer and specificity in the serum were detected by ELISA.
  • mice 6-8 weeks old Harbour human antibody transgenic mice (purchased from Beijing Viton Lihua Company) were used for immunization. The mice were raised under SPF conditions. All mice were immunized 3-4 times with a gene gun through the abdomen, each time 3-4 shots, 1.0 ⁇ g cDNA amount per shot. The interval between the initial immunization and the first booster immunization is 2 weeks, and the interval between subsequent immunizations is 3 weeks. Blood was collected 7 days after each booster immunization, and the antibody titer in serum was detected by ELISA.
  • Table 3 and Figure 3 are the results of TIM-3-hFC protein immune serum, antibody titer detection by ELISA.
  • mice after immunization with immunogen, the ELISA titer of most mice can reach more than 1:1000 after 3 immunizations, indicating that mice have a good humoral immune response to the immunogen, and their spleen cells can be used for hybridization Preparation of tumor cells.
  • mice with titers that meet the requirements can choose to perform cell fusion and hybridoma preparation.
  • the mice Prior to cell fusion, the mice were injected intraperitoneally with 50-100 micrograms of purified TIM-3-hFc per mouse for the last immunization. After 3-5 days, the mice were sacrificed and spleen cells were collected. Wash the cells by centrifugation with DMEM basal medium at 1000 rpm for 3 times, and then mix with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 5:1 viable cells, using high-efficiency electrofusion or PEG method (see METHODS IN ENZYMOLOGY, VOL. 220) for cell fusion.
  • the fused cells are diluted into DMEM medium containing 20% fetal bovine serum and 1 ⁇ HAT, and the percentage is the mass percentage. Then add 1 ⁇ 10 5 /200 microliters per well to a 96-well cell culture plate and place it in a 5% CO 2 , 37°C incubator, the percentage being a volume percentage. After 14 days, the cell fusion plate supernatant was screened by ELISA and Acumen (microplate cell detection method), and the positive clones with OD 450nm >1.0 and MFI value>100 in Acumen were amplified to 24 well plates, containing 10%( w/w) DMEM (Invitrogen) medium of fetal bovine serum was expanded at 37°C under 5% (v/v) CO 2 conditions.
  • the culture medium of the expanded culture in the 24-well plate was centrifuged, the supernatant was collected, and the supernatant was analyzed for antibody subtype.
  • the binding activity of the antibody to TIM-3 protein and TIM-3 positive cells was determined by ELISA and FACS.
  • the hybridoma cells with OD 450nm > 1.0 in the ELISA experiment and MFI value> 100 in the FACS experiment were selected as positive clones that meet the conditions.
  • DMEM obtained by cryopreservation in liquid nitrogen according to the conventional method, and can be used for subsequent Antibody production, purification and amino acid sequence determination.
  • Enzyme-linked immunosorbent assay to detect the binding of antibody to TIM-3 protein
  • the human TIM-3 protein extracellular region amino acid sequence 22-200 (as shown in the sequence listing SEQ ID NO.71) described in the step of Example 1 was cloned into a pCpC vector with human IgG Fc fragment (hFc), and transferred Transfect HEK293 cells, collect cell culture fluid, and purify to obtain the human TIM-3 protein with hFc tag (herein referred to as hTIM-3-hFc protein); the amino acid sequence of the extracellular region of monkey-derived TIM-3 protein 22-201 (such as Sequence Listing SEQ ID NO.72) cloned into pCpC vector with human IgG Fc fragment (hFc), transfected HEK293 cells, collected cell culture fluid, purified to obtain monkey TIM-3 protein with hFc tag (herein referred to as cTIM-3-hFc protein); clone the amino acid sequence 22-191 of the extracellular region of the mouse-derived TIM-3 protein (as shown in the sequence listing S
  • TIM-3 extracellular domain proteins hTIM-3-hFc, cTIM-3-hFc, mTIM-3-hFc
  • PBS Purified human, monkey, and mouse TIM-3 extracellular domain proteins
  • the pIRES plasmid containing the full-length nucleotide sequence encoding human TIM-3 as described in Example 1 was transfected into the CHOK1 cell line to obtain the CHOK1 stable transfected cell line containing human TIM-3 (herein referred to as CHOK1-hTIM-3 stable) Cell line), the pIRES plasmid with the full-length gene of monkey-derived TIM-3 is transfected into the CHOK1 cell line to construct a CHOK1 stable transfected cell line containing monkey TIM-3 (herein referred to as CHOK1-cTIM-3 stable cell line) .
  • the CHOK1-hTIM-3 stable cell line and the CHOK1-cTIM-3 stable cell line were expanded to 90% confluence in T-75 cell culture flasks, the medium was exhausted, and washed with HBSS (Hanks' Balanced Salt Solution) 1 -2 times, then treat and collect the cells with enzyme-free cell dissociation solution (Versene solution: Life technology). Wash cells with HBSS buffer 1-2 times, after cell counting, dilute cells with HBSS to 1-2x10 6 cells per ml, add 1% goat serum blocking solution, incubate on ice for 20-30 minutes, and then wash with HBSS centrifugation 2 times.
  • HBSS Hors' Balanced Salt Solution
  • the collected cells were suspended in FACS buffer (HBSS + 1% BSA) to 2x10 6 cells / ml, 100 [mu] l per well were added to 96-well FACS reaction plate, antibody sample to be tested was added 100 microliters per well of ice Incubate for 1-2 hours. Wash twice by centrifugation with FACS buffer, add 100 microliters of fluorescent (Alexa 488) labeled secondary antibody per well, and incubate on ice for 0.5-1.0 hours. The cells were centrifugally washed with FACS buffer 2-3 times, 100 ⁇ l of fixative solution (4% Paraformaldehyde) per well was added to suspend cells, and after 5-10 minutes, the cells were centrifugally washed with FACS buffer 1-2 times.
  • FACS buffer HBSS + 1% BSA
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACSCalibur, BD).
  • FACS Fluorescence Activated Cell Sorting
  • Table 7 and Table 8 where the IgG control is rat IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • the results show that the antibody to be tested can bind to the human or monkey TIM-3 protein on the cell surface.
  • Lymphocyte stimulation test detected that TIM-3 antibody blocked the binding of TIM-3 protein to its receptor to release its inhibition of T lymphocyte activity, thereby stimulating T cell proliferation.
  • the volume ratio of Ficoll to diluted whole blood is 3:4, avoid shaking and mixing, centrifuge at 400g at room temperature and 20°C for 30 minutes, the centrifuge tube is divided into three layers, the upper layer is plasma, and the middle milky white layer That is mononuclear lymphocytes.
  • a sterile pipette to gently aspirate the middle layer cells, collect into a new centrifuge tube, dilute to three times the volume with PBS phosphate buffer, centrifuge at 100g for 10 minutes at room temperature, and discard the supernatant. Resuspend the lymphocytes to 10 mL with PBS phosphate buffer and repeat the previous steps to remove the platelets. Finally, the lymphocytes were resuspended to 10 mL of a multi-component RPMI1640 medium (purchased from Invitrogen) containing 10% fetal bovine serum for use, namely peripheral blood mononuclear lymphocyte PBMC, and the percentage was the mass percentage.
  • RPMI1640 medium purchased from Invitrogen
  • OKT3 antibody (eBioscience Cat#16-0037-81) was diluted with PBS to a final concentration of 1.0 ⁇ g/ml, and then added to 6-well cell culture plates at 2 ml per well. Sealed with plastic film, incubated at 4°C overnight, washed three times with PBS the next day, inoculated the isolated PBMC cells into a 6-well plate, and cultured at 37°C, 5% CO 2 incubator for 72 hours.
  • a single chain variable fragment (scFv) of anti-human CD3 monoclonal antibody OKT3 was fused to the mouse CD8a C-terminal domain (113-220) to construct a T cell conjugate, a membrane-mismatched chimeric antibody (OS8), the mouse CD8a
  • the C-terminal domain includes the chain, transmembrane and cytoplasmic domains, so that the anti-CD3scFv fragment can be mislocated to the cell surface as a T cell activator.
  • CHOK1 cell line was transfected with a plasmid expressing the recombinant fusion protein OS8 to obtain a CHOK1 stable transfected cell line (herein referred to as CHOK1-OS8 stable cell line) expressing OS8 (a T cell activation molecule) on the cell surface.
  • CHOK1-OS8 stable cell line a CHOK1 stable transfected cell line expressing OS8 (a T cell activation molecule) on the cell surface.
  • CHOK1-OS8 cells were washed with 10 ⁇ g/mL mitomycin at 37°C for 2 hours and washed 3 times with PBS to remove residual mitomycin.
  • the pre-stimulated peripheral blood mononuclear lymphocyte PBMC was plated into 96-well cell culture plates at 1 ⁇ 10 5 cells and 100 ⁇ l per well, and then the sample solution to be tested was added to the culture plate and incubated at room temperature for 30 minutes. Finally, CHOK1-OS8 cells were added, and 2.5 ⁇ 10 4 cells of 50 ⁇ l were plated into 96-well cell culture plates to ensure a volume of 200 ⁇ L per reaction well. The reaction plates were incubated at 37°C and 5% CO 2 incubator for 20 The supernatant was collected after hours to obtain the cell supernatant, which was frozen at -20°C, and the percentage was a volume percentage.
  • the cytokine interferon IFN- ⁇ enzyme-linked immunosorbent assay in the cell supernatant uses the R&D system related detection kit human IFN- ⁇ DuoSet ELISA (DY285) and operates according to the instructions. All detection reagents except the detection antibody are provided by the detection kit.
  • the double-antibody sandwich ELISA kit (purchased from R&D Systems, IFN- ⁇ Cat#DY285) was used for enzyme-linked immunosorbent assay to determine the content of cytokine IFN- ⁇ in the cell supernatant.
  • the experimental operation is strictly in accordance with the requirements of the kit instructions, and all detection reagents are provided by the kit.
  • the specific experiment is briefly described as follows: IFN- ⁇ polyclonal antibody is coated on the ELISA microplate, sealed with plastic film and incubated at 4°C overnight, the plate is washed 4 times with the plate washing solution the next day, and the blocking solution is added to block at room temperature to block 1 -2 hours.
  • wash the plate 4 times with the plate washing solution use the cell supernatant obtained in step 4 as the test sample, add the standard and the test sample and incubate at room temperature for 2 hours.
  • the effect of TIM-3 antibody on IFN- ⁇ secretion in the PBMC stimulation experiment was examined.
  • the results are shown in Figure 9 and Table 9.
  • the IgG control is rat IgG, and the data in the table are IFN- ⁇ values (pg/mL).
  • the results show that the antibody to be tested in the PBMC lymphocyte stimulation test can enhance the IFN- ⁇ secretion of PBMC.
  • RNA isolation After the supernatant obtained from the subcloning culture of Example 1 was tested for antigen binding (that is, after the assay and activity measurement of Example 2), 5 ⁇ 10 7 hybridoma cells were collected by centrifugation, and 1 mL of Trizol was added Mix and transfer to a 1.5mL centrifuge tube and let stand at room temperature for 5 minutes. Add 0.2mL of chloroform, shake for 15 seconds, let stand for 2 minutes and centrifuge at 12,000g for 5 minutes at 4°C. Take the supernatant and transfer to a new 1.5mL centrifuge tube.
  • Reverse transcription and PCR Take 1 ⁇ g of total RNA and configure 20 ⁇ l system. After adding reverse transcriptase, react at 42°C for 60 minutes and at 7°C for 10 minutes to terminate the reaction. Configure 50 ⁇ l PCR system, including 1 ⁇ l cDNA, 25pmol of each primer, 1 ⁇ l DNA polymerase and matching buffer system, 250 ⁇ mol dNTPs. The PCR program was set, pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 35 seconds, and extension at 72°C for 5 minutes after 35 cycles to obtain PCR products.
  • the kit used for reverse transcription is PrimeScript RT Master, purchased from Takara, catalog number RR036; the kit used for PCR is Q5 ultra-fidelity enzyme, purchased from NEB, catalog number M0492.
  • Cloning and sequencing take 5 ⁇ l PCR product for agarose gel electrophoresis detection, and use the column recovery kit to purify the positive test samples, of which the recovery kit is Gel & PCR Clean-up, purchased from MACHEREY-NAGEL, Catalog No. 740609.
  • Perform ligation reaction sample 50ng, T vector 50ng, ligase 0.5 ⁇ l, buffer 1 ⁇ l, reaction system 10 ⁇ l, and ligation product at 16°C for half an hour.
  • the ligation kit is T4 DNA ligase, purchased from NEB, article number M0402; Take 5 ⁇ l of the ligation product and add it to 100 ⁇ l of competent cells (Ecos 101 competent cells, purchased from Yeastern, Catalog No.
  • sequencing results are shown in the appendix sequence information of the present invention, and the sequence information of the seven cloned heavy chain CDR1-3 and light chain CDR1-3 are shown in Table 18.
  • Example 2 has obtained purified TIM-3 antibody from the culture supernatant of hybridoma cells, and according to the sequencing results of Example 3, it is clear that the heavy chain of TIM-3 antibody is variable Region and light chain variable region sequences.
  • the heavy chain variable region sequence of the TIM-3 antibody was recombined into an expression vector containing a signal peptide and a human heavy chain antibody IgG4 constant region (where the expression vector was purchased from Invitrogen), and the light chain variable region of the TIM-3 antibody
  • the sequence was recombined into an expression vector containing the signal peptide and the human antibody light chain kappa constant region, and the recombinant plasmid was obtained and verified by sequencing (the sequencing method is the same as the sequencing method in Example 3).
  • the recombinant plasmid with medium purity and mass of 500 ⁇ g or more was filtered through 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • 293E cells purchased from Invitrogen
  • Freestyle 293 expression medium purchased from Invitrogen.
  • the shaker was set at 37°C, 130 RPM, and 8% CO 2 (v/v) concentration.
  • Freestyle 293 expression medium added 10% (v/v) F68 (purchased from Invitrogen) to a final F68 concentration of 0.1% (v/v) at the time of transfection.
  • the antibody titer of the culture broth was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes) and filtered through a 0.22 ⁇ m filter membrane to obtain the filtered cell supernatant for purification.
  • Enzyme-linked immunosorbent assay to detect the binding of antibody to TIM-3 protein
  • the results are shown in Figure 10, Figure 11 and Figure 12, Table 10, Table 11 and Table 12.
  • the IgG control is human IgG, and the data in the table are A450nm values.
  • the results are shown in Figures 13 and 14, Table 13 and Table 14.
  • the IgG control is human IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • TIM-3 receptor ligand binding experiment detects that TIM-3 antibody blocks the binding of TIM-3 to its ligand phosphatidylserine
  • hTIM-3-hFc strongly binds to staurosporine-treated Jurkat cells, but does not bind to untreated Jurkat cells. Wash the cells 1-2 times with PBS buffer. After counting the cells, dilute the cells with binding buffer to 1-2 ⁇ 10 6 cells per ml. Add 100 ⁇ l per well to the 96-well FACS reaction plate. Configure the antibody sample to be tested with 2 ⁇ g/ml hTIM-3-hFc protein with binding buffer, and mix in equal volume.
  • Table 15 Full human TIM-3 antibody blocks the binding of TIM-3 protein to its receptor phosphatidylserine
  • Lymphocyte stimulation test detected that TIM-3 antibody blocked the binding of TIM-3 protein to its receptor to release its inhibition of T lymphocyte activity, thereby stimulating T cell proliferation.
  • the results are shown in Figure 16 and Figure 17, Table 16 and Table 17.
  • the PBMC cells of two donors were selected in this experiment, and the results were basically consistent.
  • the hIgG control is human IgG, and the data in the table are IFN- ⁇ values (pg/mL).
  • the results show that the antibody to be tested in the PBMC lymphocyte stimulation test can increase the IFN- ⁇ secretion of PBMC to varying degrees.
  • the AHC sensor select the AHC sensor, and equilibrate the sensor with buffer for 10 min, then dilute the antibody to be tested to 5 ⁇ g/ml with buffer, solidify the antibody with the sensor for 3-5 min, and the signal value is 1-2 nm in height.
  • the sensor was equilibrated with buffer for 3 min, and then the antigen protein was diluted to 100 nM (the highest concentration was tentatively 100 nM) to bind and dissociate the antibody conjugated to the sensor. If a sufficient signal value is obtained, then the antigen protein is diluted by several concentration gradients to analyze the binding and dissociation of antibody antigen.
  • the sensor surface was regenerated with 10 mM Glycine, pH 1.5.
  • the kinetic rate constant needs to be subtracted from the blank control, and the global fit analysis method 1:1 is combined with the model to perform data fitting.
  • monoclonal antibodies are one of the most successful and transformative treatments in cancer treatment in the past 20 years. Compared with traditional chemical drugs, antibody drugs have higher specificity and lower toxicity. Although monoclonal antibodies have achieved continuous success, they still face many challenges.
  • mouse monoclonal antibodies have greater limitations in the diagnosis and treatment of diseases such as tumors and organ transplants; chimeric antibodies still retain 30% of the mouse-derived sequences, which can cause different levels of HAMA responses.
  • different chimeric antibodies have different degrees of immunogenicity; humanized antibodies are also called transplanted antibodies.
  • Simple CDR transplantation often leads to a decrease in the affinity of antigens and antibodies. Because it still has at least 10% of heterologous proteins, it is still restricted to varying degrees in clinical application. Therefore, it is necessary to further develop a more complete therapeutic antibody-fully human antibody.
  • H2L2 transgenic mouse is a transgenic mouse technology developed by the laboratory of Professor Frank Grosveld of the Rotterdam University Medical Center (Rotterdam, Netherlands) licensed by Heplatin Medicine (Shanghai) Co., Ltd.
  • the produced antibody has mature affinity, the variable region is fully humanized, and has excellent solubility.
  • Genetically engineered mouse technology is one of the main tools for producing fully human antibodies.
  • Cancer immunotherapy refers to the treatment of cancer through the immune system. Recently, cancer immunotherapy has attracted much attention. In addition to surgery, chemotherapy and radiotherapy, it has become a new method of cancer treatment.
  • Immune checkpoints refer to some inhibitory signaling pathways present in the immune system, which avoids tissue damage by regulating the persistence and strength of immune responses in peripheral tissues, and participates in maintaining tolerance to self-antigens. The use of immune checkpoint inhibitory signaling pathways to inhibit T cell activity is an important mechanism for tumors to escape immune killing. Blocking immune checkpoints is one of many effective strategies for activating anti-tumor immunity.
  • Inhibitors of immune checkpoint proteins have the potential to treat various tumor types (such as metastatic melanoma, lung cancer, breast cancer, renal cell carcinoma, etc.). Recent studies on cancer immunotherapy methods have shown promising results, especially for metastatic cancer cases. In addition, cancer immunotherapy has great potential in the treatment of blood cancers, including Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, etc. The side effects caused by immune checkpoint inhibitors are negligible, reversible and controllable. Effective immune checkpoint inhibitors can significantly improve the overall survival of cancer patients. Immune checkpoint inhibitors can also be used in combination with targeted therapy or conventional radiation therapy and chemotherapy, and this combination therapy can effectively treat many types of cancer, and may be a hope for treating or curing many types of cancer.
  • TIM-3 antibody is mainly used for the treatment of malignant solid tumors and lymphomas, and it is also mainly focused on the combined use of it and other therapies or target drugs to develop antibodies with wide indications to expand its applicable clinical symptoms , Including unresectable metastatic melanoma, advanced solid cancer, breast cancer, endometrial cancer, ovarian cancer, renal cancer, pancreatic cancer, recurrent glioblastoma, head and neck cancer, bladder cancer, metastatic rectal cancer , Gastrointestinal stromal tumors, acinar cell carcinoma, advanced malignant solid tumors, non-small cell lung cancer, etc.
  • Therapeutic monoclonal antibodies can be developed by various technologies and approaches, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the preparation of monoclonal antibodies from wild-type or transgenic mice through hybridoma technology is still the mainstream of current therapeutic monoclonal antibody preparation methods.
  • the present invention adopts optimized hybridoma technology to prepare the required anti-TIM-3 antibody.
  • Antibodies can be obtained by immunizing wild-type mice, but humanized antibodies need to be humanized to obtain humanized antibodies.
  • the disadvantage is that the modified antibodies may be more immunogenic and the antibody structure may change. Resulting in loss of activity or poor productivity.
  • Fully human antibodies can be obtained by immunizing fully human transgenic mice, but the amount or affinity of the antibodies obtained will be poor.
  • the antibody expression and activity screening can be performed by constructing an antibody library of immunized mice, but the random recombination of the heavy and light chains of antibodies will lead to poor productivity of the formed antibodies.
  • the phage display technology can be used for expression and activity screening of antibodies by constructing a human-derived antibody library. However, since the antibody is not immunized, the affinity of the resulting antibody will be poor.
  • sequence information of the present invention (including the amino acid sequence of the CDR region of the TIM-3 antibody of the present invention and its sequence number, the amino acid and gene sequences of the heavy/light chain variable region of the TIM-3 antibody of the present invention and its sequence number) are shown in the table below 18
  • VH-CDR1 is a heavy chain variable region-CDR1
  • VH-CDR2 is a heavy chain variable region-CDR2
  • VH-CDR3 is a heavy chain variable region-CDR3
  • VL-CDR1 is a light chain variable region- CDR1
  • VL-CDR2 are light chain variable regions-CDR2
  • VL-CDR3 are light chain variable regions-CDR3.
  • VH-aa refers to the heavy chain variable region amino acid sequence
  • VH-nt refers to the heavy chain variable region gene sequence
  • VL-aa refers to the light chain variable region amino acid sequence
  • VL-nt refers to the heavy chain variable Region gene sequence.

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Abstract

提供了一种高特异性靶向TIM-3的高亲和力全人单克隆抗体及其制备方法，所述单抗具有显著的抗肿瘤活性，可用于制备相关的诊断试剂或者预防或治疗与TIM-3功能异常相关的疾病的药物组合物。

Description

抗人TIM-3单克隆抗体及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种TIM-3抗体及其制备方法和应用
背景技术
T细胞免疫球蛋白结构域和粘蛋白结构域3(TIM-3，HAVCR2)是一种有301个氨基酸的I型膜蛋白，是已知的主要免疫检查点(Immune Checkpoint)之一。TIM-3主要表达在活化的CD4 +和CD8 +T淋巴细胞，天然的调节性T细胞、NK细胞以及先天细胞(巨噬细胞、单核细胞和树突细胞)。据文献报道，TIM-3的配体包括磷脂酰丝氨酸(PtdSer)、半乳凝素-9(Gal-9)、高迁移率族蛋白1(HMGB1)和癌胚抗原细胞粘附分子1(CECAM1)。TIM-3通过与其配体的结合在调节免疫应答的各方面发挥作用。TIM-3与CECAM1的相互作用，抑制T细胞活化，促使T细胞耐受和耗竭。调节性T细胞和许多肿瘤细胞表达Gal-9蛋白，与TIM-3结合抑制效应T细胞的活化。HMGB1与TIM-3结合作用于抗原递呈细胞和先天细胞，减少损伤相关分子模式介导的炎症反应。TIM-3结合磷脂酰丝氨酸促进凋亡细胞的清除。
研究表明TIM-3表达的异常与许多疾病有密切的关系。有研究发现HIV，HCV感染的患者以及一些肿瘤患者，其T细胞TIM-3表达升高，介导T效应细胞的凋亡，传递负调节信号，从而导致免疫系统的紊乱，机体免疫功能瘫痪。另外，在多种小鼠肿瘤模型中的肿瘤浸润的淋巴细胞共表达TIM-3和PD-1，并且表现出功能的耗竭，丧失增殖以及分泌一些细胞因子(IL-2、TNF-α和IFN-γ)的能力。同时阻断PD-1与TIM-3信号通路相比单独阻断PD-1或TIM-3的信号通路，能更有效地抑制肿瘤生长。在人类很多肿瘤浸润的T细胞中，TIM-3 +/PD-1 +双阳性的CD8 +T细胞比TIM-3 -/PD-1 +和TIM-3 -/PD-1 -的CD8 +T细胞功能更加紊乱，分泌细胞因子的能力也更弱。
目前国内外仅有两个TIM-3抗体处于临床研究阶段，其他抗体药物仍处于发现和研究阶段，尚没有TIM-3抗体进入临床应用，急需开发出活性好、适应症广和产量高的TIM-3抗体。亟待开发活性更好的TIM-3抗体，进一步以提高治疗效果。抗体本身的活性受可变区序列和恒定区结构的影响。抗体的可变区序列决定了识别抗原的决定簇、结合亲和力及在体内代谢速率，都会影响其体内活性，甚至不同病患个人的临床效果。
目前TIM-3抗体的临床研究阶段主要用于恶性实体瘤和淋巴瘤的治疗，而且也主要集中于它和其他疗法或靶点药物的联合使用，开发适应症广的抗体从而扩大其适用的临床症状，亟待开发产量更高的TIM-3抗体以降低患者的治疗 成本，惠及更多患者。此外，目前肿瘤免疫治疗价格昂贵，急需发明并且生产新的抗体以降低成本。
发明内容
为了克服目前缺少全人TIM-3抗体及现有TIM-3抗体在活性方面的缺点，本发明提供一种亲和力高、特异性强的TIM-3抗体及其制备方法和用途。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，和
SEQ ID NO.10n+5所示的VH-CDR3；
其中，各n独立地为0、1、2、3、4、5或6；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.10n+1所示的氨基酸序列，其中，n为0、1、2、3、4、5或6。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明的第一方面所述的重链可变区。
在另一优选例中，所述重链还包括重链恒定区。
在另一优选例中，所述重链恒定区为人源。
在另一优选例中，所述重链恒定区为人源抗体重链IgG4恒定区。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
其中，各n独立地为0、1、2、3、4、5或6；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.10n+6所示的氨基酸序列，其中，n为0、1、2、3、4、5或6。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明的第三方面所述的轻链可变区。
在另一优选例中，所述轻链还包括轻链恒定区。
在另一优选例中，所述轻链恒定区为人源。
在另一优选例中，所述轻链恒定区为人源抗体轻链kappa恒定区。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明的第一方面所述的重链可变区；和/或
(2)如本发明的第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明的第二方面所述的重链；和/或如本发明的第四方面所述的轻链，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
在另一优选例中，上述任一CDR的氨基酸序列中包含经过添加、缺失、修饰和/或取代1、2或3个氨基酸的衍生CDR序列，并且使得含有所述衍生CDR序列的VH和VL所构成的衍生抗体能够保留与TIM-3结合的亲和力。
在另一优选例中，所述的衍生抗体与TIM-3结合的亲和力F1与相应非衍生的抗体与TIM-3结合的亲和力F0之比(F1/F0)为0.5-2，较佳地为0.7-1.5，和更佳地0.8-1.2。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列为同源性或序列相同性为至少96％的氨基酸序列。
在另一优选例中，所述的抗体还包括重链恒定区和/或轻链恒定区。
在另一优选例中，所述的重链恒定区为人源的，和/或所述的轻链恒定区为人源的。
在另一优选例中，所述重链恒定区为人源抗体重链IgG4恒定区，且所述轻链恒定区为人源抗体轻链kappa恒定区。
在另一优选例中，所述抗体选自下组：动物源抗体、嵌合抗体、人源化抗体、全人抗体、或其组合。
在另一优选例中，所述的嵌合抗体在人中的免疫原性Z1与非嵌合的抗体(如鼠源抗体)在人中的免疫原性Z0之比(Z1/Z0)为0-0.5，较佳地0-0.2，更佳地0-0.05(如0.001-0.05)。
在另一优选例中，所述的抗体是部分或全人源化、或全人的单克隆抗体。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述抗体为抗体全长蛋白、或抗原结合片段。
在另一优选例中，所述抗体为双特异性抗体、或多特异性抗体。
在另一优选例中，所述抗体具有选自下组的一个或多个特性：
(a)抑制肿瘤细胞迁移或转移；
(b)抑制肿瘤生长。
在另一优选例中，所述的抗体具有如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区；
其中，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
Figure PCTCN2019122471-appb-000001
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
在另一优选例中，所述的抗体具有如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区；其中，
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.3所示的VH-CDR1，
SEQ ID NO.4所示的VH-CDR2，和
SEQ ID NO.5所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.8所示的VL-CDR1，
SEQ ID NO.9所示的VL-CDR2，和
SEQ ID NO.10所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.13所示的VH-CDR1，
SEQ ID NO.14所示的VH-CDR2，和
SEQ ID NO.15所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.18所示的VL-CDR1，
SEQ ID NO.19所示的VL-CDR2，和
SEQ ID NO.20所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.23所示的VH-CDR1，
SEQ ID NO.24所示的VH-CDR2，和
SEQ ID NO.25所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.28所示的VL-CDR1，
SEQ ID NO.29所示的VL-CDR2，和
SEQ ID NO.30所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.33所示的VH-CDR1，
SEQ ID NO.34所示的VH-CDR2，和
SEQ ID NO.35所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.38所示的VL-CDR1，
SEQ ID NO.39所示的VL-CDR2，和
SEQ ID NO.40所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.43所示的VH-CDR1，
SEQ ID NO.44所示的VH-CDR2，和
SEQ ID NO.45所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.48所示的VL-CDR1，
SEQ ID NO.49所示的VL-CDR2，和
SEQ ID NO.50所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.53所示的VH-CDR1，
SEQ ID NO.54所示的VH-CDR2，和
SEQ ID NO.55所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.58所示的VL-CDR1，
SEQ ID NO.59所示的VL-CDR2，和
SEQ ID NO.60所示的VL-CDR3；
或
所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.63所示的VH-CDR1，
SEQ ID NO.64所示的VH-CDR2，和
SEQ ID NO.65所示的VH-CDR3；
所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.68所示的VL-CDR1，
SEQ ID NO.69所示的VL-CDR2，和
SEQ ID NO.70所示的VL-CDR3。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51或61所示的氨基酸序列；和/或所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56或66所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.6所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.11所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.16所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.21所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.26所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.31所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.36所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.41所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.46所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.51所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.56所示的氨基酸序列。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.61所示的氨基酸序列；且所述抗体的轻链可变区含有SEQ ID NO.66所示的氨基酸序列。
在另一优选例中，所述的抗体选自下组：
抗体编号 克隆 VH序列编号 VL序列编号
1 7A4F10 1 6
2 18D2H2 11 16
3 134H3G6 21 26
4 215A8F2 31 36
5 34B6D8 41 46
6 39E5H1 51 56
7 57F4E5 61 66。
在另一优选例中，所述重链可变区的氨基酸序列与如序列表中SEQ ID NO.1、 11、21、31、41、51或61所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述轻链可变区的氨基酸序列与如序列表中SEQ ID NO.6、16、26、36、46、56或66所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白包括：
(i)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在另一优选例中，所述重组蛋白包括：
(i)选自下组的抗体，
抗体编号 克隆 VH序列编号 VL序列编号
1 7A4F10 1 6
2 18D2H2 11 16
3 134H3G6 21 26
4 215A8F2 31 36
5 34B6D8 41 46
6 39E5H1 51 56
7 57F4E5 61 66
以及
(ii)任选的协助表达和/或纯化的标签序列。
在本发明的第七方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的多肽：
(1)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体；以及
(2)如本发明的第六方面所述的重组蛋白。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO.2、12、22、32、42、52或62所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO.7、17、27、37、47、57或67所示。
在另一优选例中，编码所述重链可变区序列的多核苷酸和编码所述轻链可 变区序列的多核苷酸选自下组：
Figure PCTCN2019122471-appb-000002
在本发明的第八方面，提供了一种载体，所述载体含有本发明的第七方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第九方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有本发明的第八方面所述的载体或基因组中整合有本发明的第七方面所述的多核苷酸。
在本发明的第十方面，提供了一种抗体偶联物，该抗体偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体、或其组合；和(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十一方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有本发明的第五方面所述的抗体。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十二方面，提供了一种药物组合物，所述药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物包括0.01～99.99％的如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
在本发明的第十三方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合，其中所述活性成分被用于(a)制备诊断试剂或试剂盒；和/或(b)制备预防和/或治疗与TIM-3表达或功能异常相关的疾病的药物。
在另一优选例中，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述TIM-3表达或功能异常相关的疾病选自下组：肿瘤和自身免疫性疾病。
在另一优选例中，所述肿瘤选自下组：黑色素瘤，间皮瘤，非小细胞肺癌，乳腺癌，肝癌，滑膜肉瘤，转移性结肠癌，肾癌，膀胱癌，前列腺癌，卵巢癌，丙型肝炎病毒慢性感染，晚期实体癌，消化器官恶性肿瘤，子宫内膜癌，复发黑色素瘤，头颈部鳞状细胞癌，皮肤T细胞淋巴瘤，输卵管癌，腹膜肿瘤，肌肉浸润性膀胱癌，广泛的阶段小细胞肺癌，成人急性髓细胞性白血病，非典型慢性粒细胞白血病，卵巢上皮细胞癌，B细胞慢性淋巴细胞白血病，皮肤B细胞非霍奇金淋巴瘤，眼内淋巴瘤，睾丸绒毛膜癌，神经母细胞瘤，食管癌。
在另一优选例中，所述自身免疫性疾病选自下组：系统性红斑狼疮、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、寻常天皰疮、类天皰疮、移植排斥。
在另一优选例中，所述诊断试剂或试剂盒用于：检测样品中TIM-3蛋白。
在另一优选例中，所述的诊断试剂或试剂盒用于诊断TIM-3相关疾病。
在另一优选例中，所述的诊断试剂或试剂盒用于检测样品中TIM-3蛋白。
在本发明的第十四方面，提供了一种体外检测(包括诊断性或非诊断性)样品中TIM-3蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在TIM-3 蛋白。
在本发明的第十五方面，提供了一种体外检测样品中TIM-3蛋白的组合物，其包括如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合作为活性成分。
在本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明的第五方面所述的抗体、如本发明的第六方面所述的重组蛋白、如本发明的第十方面所述的抗体偶联物、本发明的第十一方面所述的免疫细胞、或其组合。
在本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有本发明的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明抗体的二抗；
或者，
所述试剂盒含有如本发明的第十六方面所述的检测板。
在本发明的第十八方面，提供了一种重组多肽的制备方法，该方法包括：
(a)在适合表达的条件下，培养如本发明的第九方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明的第五方面所述的抗体或如本发明的第六方面所述的重组蛋白。
在本发明的第十九方面，提供了一种药物组合，包括：
(i)第一活性成分，所述第一活性成分包括如本发明的第五方面所述的抗体1、或如本发明的第六方面所述的重组蛋白、或如本发明的第十方面所述的抗体偶联物、或如本发明的第十一方面所述的免疫细胞、或如本发明的第十二方面所述的药物组合物、或其组合；
(ii)第二活性成分，所述第二活性成分包括第二抗体、或化疗剂。
在另一优选例中，所述第二抗体选自下组：CTLA4抗体、PD-1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
在另一优选例中，所述化疗剂选自下组：多西他赛、卡铂、或其组合。
在本发明的第二十方面，提供了本发明的第五方面所述的抗体，或本发明的第六方面所述的重组蛋白、或本发明的第十方面所述的抗体偶联物、或本发明的第十一方面所述的免疫细胞、和/或本发明的第十二方面所述的药物组合物与第二抗体或化疗剂的组合在制备用于治疗TIM-3表达或功能异常相关的疾病的药物中的用途。
在另一优选例中，所述第二抗体选自下组：CTLA4抗体、PD-1抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
在本发明的第二十一方面，提供了一种治疗与TIM-3表达或功能异常相关 的疾病的方法，向有需要的对象施用有效量的如本发明的第五方面所述的抗体、或如本发明的第六方面所述的重组蛋白、或如本发明的第十方面所述的抗体偶联物、或如本发明的第十一方面所述的免疫细胞、或如本发明的第十二方面所述的药物组合物、或其组合。
在另一优选例中，所述与TIM-3表达或功能异常相关的疾病为癌症。
在另一优选例中，所述的方法还包括：在施用第一活性成分之前、之中和/或之后，向所述对象施用安全有效量的第二抗体。
在另一优选例中，所述的第二抗体选自下组：PD-1抗体、CTLA4抗体。
在另一优选例中，所述的第二抗体为PD-1抗体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了TIM-3-hFc蛋白与其商业化抗体的结合活性。
图2显示了TIM-3基因转染的HEK293细胞FACS检测结果。
图3显示了ELISA检测TIM-3-hFC蛋白免疫后小鼠血清抗体效价。
图4显示了酶联免疫吸附实验中TIM-3抗体与人TIM-3胞外区蛋白反应活性。
图5显示了酶联免疫吸附实验中TIM-3抗体与猴TIM-3胞外区蛋白反应活性。
图6显示了酶联免疫吸附实验中TIM-3抗体与鼠TIM-3胞外区蛋白反应活性。
图7显示了FACS检测TIM-3抗体与CHOK1-hTIM-3的结合反应。
图8显示了FACS检测TIM-3抗体与CHOK1-cTIM-3的结合反应。
图9显示了抗体在OKT3依赖的PBMC激活试验中对IFN-γ分泌的影响。
图10显示了酶联免疫吸附实验中全人TIM-3抗体与人TIM-3胞外区蛋白反应活性。
图11显示了酶联免疫吸附实验中全人TIM-3抗体与猴TIM-3胞外区蛋白反应活性。
图12显示了酶联免疫吸附实验中全人TIM-3抗体与鼠TIM-3胞外区蛋白反应活性。
图13显示了FACS检测全人TIM-3抗体与CHOK1-hTIM-3的结合反应。
图14显示了FACS检测全人TIM-3抗体与CHOK1-cTIM-3的结合反应。
图15显示了全人TIM-3抗体阻断TIM-3蛋白与其受体磷脂酰丝氨酸的结合。
图16显示了全人TIM-3抗体在淋巴细胞激活试验中对IFN-γ分泌的影响(捐赠人X)。
图17显示了全人TIM-3抗体在淋巴细胞激活试验中对IFN-γ分泌的影响(捐赠人Y)。
具体实施方式
本发明人通过广泛而深入的研究，以人源TIM-3作为免疫原，采用优化的杂交瘤技术制备TIM-3抗体。具体而言，本发明采用人源抗体转基因小鼠技术进行全人源抗体的制备，获得TIM-3抗体的先导抗体；再通过对先导抗体的初步生产、纯化和检定，获得具备抗体亲和力高、在人外周血单核细胞激活反应中显著增加IFN的表达水平等优异生物性特性的TIM-3抗体；然后通过分子生物学方法测序获知TIM-3抗体的重链可变区和轻链可变区的氨基酸序列。所述的TIM-3抗体与人源和猴源的TIM-3蛋白均具有高度亲和力，能在OKT3激活诱导的人外周血单核细胞中增加IFN的表达水平。本发明还提供了这些抗体的用途，包括但不仅限于抑制TIM-3与其配体介导的信号通路的负调控，激活肿瘤特异免疫反应，单独或与抗PD-1，CTLA-4单克隆抗体或其它抗肿瘤药物联合应用于用于肿瘤免疫治疗。在此基础上完成了本发明。
术语
本发明中，“VH-CDR1”与“CDR-H1”可互换使用，均指重链可变区的CDR1；“VH-CDR2”与“CDR-H2”可互换使用，均指重链可变区的CDR2；“VH-CDR3”与“CDR-H3”可互换使用，均指重链可变区的CDR3。“VL-CDR1”与“CDR-L1”可互换使用，均指轻链可变区的CDR1；“VL-CDR2”与“CDR-L2”可互换使用，均指轻链可变区的CDR2；“VL-CDR3”与“CDR-L3”可互换使用，均指轻链可变区的CDR3。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不 均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表19进行氨基酸替换而产生。
表19
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗TIM-3的抗体
本发明中，所述抗体为抗TIM-3的抗体。本发明提供一种针对TIM-3的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，
所述的重链可变区(VH)具有选自下组的互补决定区CDR：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，和
SEQ ID NO.10n+5所示的VH-CDR3；
其中，各n独立地为0、1、2、3、4、5或6；
所述的轻链可变区(VL)具有选自下组的互补决定区CDR：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
其中，各n独立地为0、1、2、3、4、5或6；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
优选地，重链可变区(VH)包括以下三个互补决定区CDR：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，和
SEQ ID NO.10n+5所示的VH-CDR3；
轻链可变区(VL)包括以下三个互补决定区CDR：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
各n独立地为0、1、2、3、4、5或6；较佳地n为0或1；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。 公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
本发明中的抗体可以是全长蛋白(如IgG1,IgG2a,IgG2b或者IgG2c)，也可以是包含抗原抗体结合域的蛋白片段(例如Fab，F(ab’),sdAb,ScFv片段)。本发明中的抗体可以是野生型蛋白，也可以是经过特定突变已达到某种特定效果的突变型蛋白，例如利用突变消除抗体的效应子功能。
较佳地，本文所述抗体为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向TIM-3(例如人TIM-3)的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、 51或61所示的氨基酸序列。
在另一优选例中，所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56或66所示的氨基酸序列。
在另一优选例中，所述靶向TIM-3的抗体的重链可变区(VH)氨基酸序列，和/或，轻链可变区氨基酸序列如下表20所示：
表20
抗体编号 VH序列编号 VL序列编号
1 1 6
2 11 16
3 21 26
4 31 36
5 41 46
6 51 56
7 61 66
在另一优选例中，所述靶向TIM-3的抗体为7A4F10、10D2H2、134H3G6、215A8F2、34B6D8、39E5H1、57F4E5。
在另一优选例中，所述靶向TIM-3的抗体为7A4F10。
在另一优选例中，所述靶向TIM-3的抗体为10D2H2。
重组蛋白
本发明还提供一种重组蛋白，其包括TIM-3抗体的重链CDR1(VH-CDR1)、重链CDR2(VH-CDR2)和重链CDR3(VH-CDR3)中的一种或多种，和/或，TIM-3抗体的轻链CDR1(VL-CDR1)、轻链CDR2(VL-CDR2)和轻链CDR3(VL-CDR3)中的一种或多种，
所述重链CDR1-3的序列如下：
SEQ ID NO.10n+3所示的VH-CDR1，
SEQ ID NO.10n+4所示的VH-CDR2，
SEQ ID NO.10n+5所示的VH-CDR3；
所述轻链CDR1-3的序列如下：
SEQ ID NO.10n+8所示的VL-CDR1，
SEQ ID NO.10n+9所示的VL-CDR2，和
SEQ ID NO.10n+10所示的VL-CDR3；
各n独立地为0、1、2、3、4、5或6；较佳地n为0或1；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括TIM-3抗体的重链可变区和/或TIM-3抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51或61所示的氨基酸序列；所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56或66所示的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括TIM-3抗体的重链可变区和TIM-3抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51或61所示的氨基酸序列，且所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56或66所示的氨基酸序列。
在另一优选例中，所述重组蛋白及其包括的重链CDR1-3、轻链CDR1-3的氨基酸序列的序列编号如表21所示：
表21重链CDR1-3、轻链CDR1-3的氨基酸序列的序列编号
Figure PCTCN2019122471-appb-000003
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
较佳地，所述的重组蛋白还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为大鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为大鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
所述的重组蛋白为本领域常规的蛋白质，较佳地，其为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain  antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述重组蛋白的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述重组蛋白。
核酸
本发明还提供一种核酸，其编码上述的抗体(例如抗TIM-3的抗体)或重组蛋白或抗TIM-3的抗体的重链可变区或轻链可变区。
较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO.2、12、22、32、42、52或62所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID NO.7、17、27、37、47、57或67所示。
更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO.2、12、22、32、42、52或62所示；且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID NO.7、17、27、37、47、57或67所示。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
载体
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞； 或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位 于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2019122471-appb-000004
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
应用
本发明还提供了本发明抗体、抗体偶联物ADC、重组蛋白、和/或免疫细胞的用途，例如用于制备诊断制剂或制备药物。
较佳地，所述的药物是用于预防和/或治疗与TIM-3表达或功能异常相关的疾病的药物。
本发明抗体、ADC、重组蛋白、和/或免疫细胞的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗肿瘤发生、生长和/或转移，尤其是TIM-3高表达的肿瘤。所述肿瘤包括(但并不限于)：黑色素瘤，间皮瘤，非小细胞肺癌，乳腺癌，肝癌，滑膜肉瘤，转移性结肠癌，肾癌，膀胱癌，前列腺癌，卵巢癌，丙型肝炎病毒慢性感染，晚期实体癌，消化器官恶性肿瘤，子宫内膜癌，复发黑色素瘤，头颈 部鳞状细胞癌，皮肤T细胞淋巴瘤，输卵管癌，腹膜肿瘤，肌肉浸润性膀胱癌，广泛的阶段小细胞肺癌，成人急性髓细胞性白血病，非典型慢性粒细胞白血病，卵巢上皮细胞癌，B细胞慢性淋巴细胞白血病，皮肤B细胞非霍奇金淋巴瘤，眼内淋巴瘤，睾丸绒毛膜癌，神经母细胞瘤，食管癌。
(ii)诊断、预防和/或治疗自身免疫性疾病，所述自身免疫性疾病包括(但并不限于)：系统性红斑狼疮、口眼干燥综合征、类风湿性关节炎、强直性脊柱炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、寻常天皰疮、类天皰疮、移植排斥。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明所述的药物组合物是用于预防和/或治疗与TIM-3表达或功能异常相关的疾病的药物组合物。
本发明的药物组合物可直接用于结合TIM-3蛋白分子，因而可用于预防和治疗肿瘤等疾病。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提供上述药物组合物在制备预防和/或治疗与TIM-3表达或功能异常相关的疾病的药物中的应用。较佳地，所述与TIM-3表达或功能异常相关的疾病为癌症和自身免疫性疾病。
检测样品中TIM-3蛋白的方法、组合物
本发明还提供一种检测样品中TIM-3蛋白(例如检测过表达TIM-3细胞)的方法，包括如下的步骤：上述的抗体与待检样品在体外接触，检测上述的抗体与所述待检样品是否结合形成抗原-抗体复合物即可。
所述的过表达的含义为本领域常规，指TIM-3蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表 达和功能活性提高(如在底物的酶水解作用增加的情况下)。
本发明中，上述是否结合形成抗原-抗体复合物的检测方式是本领域常规的检测方式，较佳地为流式细胞实验(FACS)检测。
本发明提供一种检测样品中TIM-3蛋白的组合物，其包括上述的抗体、重组蛋白、抗体偶联物、免疫细胞、或其组合作为活性成分。较佳地，其还包括上述的抗体的功能片段组成的化合物作为活性成分。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的主要优点在于：
(1)本发明采用大鼠-人嵌合抗体转基因小鼠获得全人抗体，所获得抗体具有一系列的优异特征：
①可变区序列与现有抗体有差异(同源性<92％)；
②所获得抗体具有较强的亲和力；
③所获得抗体具有很好的刺激T细胞激活活性；
(2)本发明采用的转基因小鼠，与野生型小鼠比能够更容易获得全人源抗体，从而降低抗体的免疫原性；与全人抗体的转基因小鼠比，获得的抗体数量多、亲和力强，序列多样性好并且活性高；
(3)本发明采用杂交瘤技术获得抗体，与噬菌体库获得的抗体相比，抗体亲和力高，序列表达良好；
(4)本发明获得了序列不同的抗体，能够与TIM-3特异性结合，其结合活性低于纳摩尔，通过逆转TIM-3对T细胞激活活性的抑制，从而激活T细胞分泌IFN。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件(例如商品说明书)。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
实施例1：TIM-3特异性抗体的制备
(一)、免疫原的制备
制备包括胞外区TIM-3蛋白、TIM-3重组细胞株、TIM-3 DNA载体的表达质粒等免疫原。
免疫原1)，将人源TIM-3蛋白胞外区氨基酸序列22-200(如序列表SEQ ID  NO.71所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293细胞(购自Invitrogen)进行瞬时转染(PEI，Polysciences)并使用FreeStyle  TM 293(Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含TIM-3蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的TIM-3蛋白(TIM-3-hFc)，用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原人TIM-3-hFc蛋白。免疫原TIM-3-hFc蛋白在使用前需要进行一系列质控检测，如检测其蛋白浓度、纯度、分子量和生物活性等。
其中，免疫原TIM-3-hFc与商业化抗TIM-3抗体的结合活性采用ELISA检测，具体为：
将带hFc标签的TIM-3蛋白(TIM-3-hFc，即免疫原)，用PBS稀释至1μg/mL，以100μl/孔加入ELISA微孔板，4℃孵育过夜。用ELISA封闭液(含1％BSA，pH7.4的PBS磷酸缓冲液，所述百分比为质量百分比)37℃封闭两小时后，再加入梯度稀释的与商业化抗TIM-3抗体，37℃温育1小时。所述商业化抗TIM-3抗体购自R&D system，商品号MAB2365。加入HRP(辣根过氧化物酶)标记的二抗，室温孵育30分钟后加入100微升/孔TMB显色液。室温孵育15分钟后，加入50微升1N盐酸终止显色反应，用ELISA读板机读取OD450nm读数。每个步骤之后都需要洗板。其中阴性对照抗体为rat IgG，结果如图1和表1所示。
表1 TIM-3-hFc蛋白与其商业化抗体的结合活性
Figure PCTCN2019122471-appb-000005
Figure PCTCN2019122471-appb-000006
从结果说明TIM-3与商业化抗TIM-3蛋白的抗体在蛋白水平的结合随抗体的浓度变化而变化。
免疫原2)，人源TIM-3全长氨基酸序列克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系和CHOK1细胞系(均购自Invitrogen)进行质粒转染(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)后，在含0.5μg/ml嘌呤霉素的含10％(w/w)FBS的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO 2培养。大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的TIM-3抗体染色，经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原TIM-3重组细胞株。具体选择结果如表2和图2所示，表2中阳性细胞(％)指阳性细胞占总细胞数目的百分比，MFI是所测细胞群的平均荧光强度值。图2说明，HEK293细胞有较高水平TIM-3的表达。
表2 TIM-3基因转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2019122471-appb-000007
免疫原3)，TIM-3全长氨基酸序列cDNA被克隆到pCDNA3.1载体并包被到1.0um金胶体子弹上，用Helios基因枪(Bio-rad)免疫。详细方法根据Helios基因枪说明书进行制定。
(二)、杂交瘤细胞的制备和抗体筛选
Harbour转基因小鼠引入了人免疫球蛋白可变区基因和大鼠免疫球蛋白恒定区基因，而小鼠本身的Ig表达则被沉默(F.G.Franklin,et al,patent#WO 2010/070263 Al)。该转基因小鼠经抗原免疫后能产生与正常小鼠(如Balb/c)相当的免疫反应和抗体效价。
A、免疫原1)免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原TIM-3-hFc蛋白用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射100微克免疫原蛋白。加强免疫时，免疫原蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原蛋白。初次免疫与第一次加强免疫之间间隔2周，以后 每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原蛋白的抗体效价和特异性，结果如图3和表3所示。
表3 ELISA检测TIM-3-hFC蛋白免疫后小鼠血清抗体效价
Figure PCTCN2019122471-appb-000008
表3说明，经TIM-3-hFc免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一万左右。其中空白对照为1％(w/w)BSA，其中批次指第二次加强免疫后第七天的小鼠血清，表中的数据为OD 450nm值。
B、免疫原2)免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。将得到的含人源TIM-3的HEK293-h TIM-3稳定细胞系在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基。用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2×10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用ELISA检测血清中抗体效价和特异性。
C、免疫原3)免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。所有小鼠经腹部用基因枪免疫3-4次，每次3-4枪，每枪1.0微克cDNA量。初次免疫与第一次加强免疫之间隔2周，以后每次免疫间隔3周。每次加强免疫7天后采血，用ELISA检测血清中抗体效价。
通常大部分小鼠经2-3次免疫后ELISA效价可达到1:1000以上。表3和图3为TIM-3-hFC蛋白免疫血清，用ELISA进行抗体效价检测的结果。
结果表明：以免疫原进行免疫，大部分小鼠经3次免疫后ELISA效价可达到1:1000以上，说明小鼠对免疫原产生较好的体液免疫反应，其脾细胞可以用来进行杂交瘤细胞制备。
效价符合要求的小鼠可选择进行细胞融合和杂交瘤制备。细胞融合前，小鼠最后一次免疫用每只50-100微克纯化的TIM-3-hFc腹腔注射。3-5天后处死小鼠，收集脾细胞。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后 按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0混合(购自ATCC)，采用高效电融合或PEG方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1×HAT的DMEM培养基中，所述百分比为质量百分比。然后按1×10 5/200微升每孔加入到96孔细胞培养板中，放入5％CO 2、37℃培养箱中，所述百分比为体积百分比。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD 450nm>1.0和Acumen中MFI值>100的阳性克隆扩增到24孔板，在含10％(w/w)胎牛血清的DMEM(Invitrogen)的培养基中，在37℃，5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析。用ELISA、FACS确定抗体对TIM-3蛋白和TIM-3阳性细胞的结合活性。
根据24孔板筛选结果，挑选ELISA实验中OD 450nm>1.0、FACS实验中MFI值>100的杂交瘤细胞为符合条件的阳性克隆。选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自Invitrogen)37℃，5％(v/v)CO 2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性评估生物活性(评估标准为ELISA实验中OD 450nm>1.0、FACS实验中MFI值>100)。
根据24孔板样品检测结果，阳性克隆在含10％(w/w)FBS的DMEM(购自Invitrogen)培养基中，在37℃，5％(v/v)CO 2条件下进行扩大培养，细胞悬浮于冻存液[含有20％(w/w)FBS和10％(w/w)DMSO的DMEM]中，按常规方法液氮冻存即得本发明杂交瘤细胞，并可用于后续的抗体生产、纯化和氨基酸序列测定。
实施例2：嵌合抗体的鉴定
(一)酶联免疫吸附实验(ELISA)检测抗体与TIM-3蛋白的结合
将实施例1步骤中所述的人源TIM-3蛋白胞外区氨基酸序列22-200(如序列表SEQ ID NO.71所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体，转染HEK293细胞，收集细胞培养液，纯化得到带hFc标签的人TIM-3蛋白(此处称为hTIM-3-hFc蛋白)；将猴源TIM-3蛋白胞外区氨基酸序列22-201(如序列表SEQ ID NO.72所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体，转染HEK293细胞，收集细胞培养液，纯化得到带hFc标签的猴TIM-3蛋白(此处称为cTIM-3-hFc蛋白)；将鼠源TIM-3蛋白胞外区氨基酸序列22-191(如序列表SEQ ID NO.73所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体，转染HEK293细胞，收集细胞培养液，纯化得到带hFc标签的鼠TIM-3蛋白(此处称为mTIM-3-hFc蛋白)。纯化的人、猴、鼠TIM-3胞外区蛋白(hTIM-3-hFc、cTIM-3-hFc、mTIM-3-hFc)用PBS稀释到终浓度1.0μg/ml,然后以100μl每孔加到96孔ELISA 板。用塑料膜封好4℃孵育过夜，第二天用洗板液(PBS+0.01％Tween20)洗板2次，加入封闭液(PBS+0.01％Tween20+1％BSA)室温封闭1-2小时。倒掉封闭液，加入待测抗体样品50-100μl每孔,37℃孵育1-2小时后，用洗板液(PBS+0.01％Tween20)洗板2-3次.加入HRP(辣根过氧化物酶)标记的二抗，37℃孵育1-2小时后，用洗板液(PBS+0.01％Tween20)洗板2-3次。加入TMB底物100μl每孔，室温孵育15-30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(TiterMax 384plus,Molecular Device)读取A450nm数值。结果如图4,图5和图6，表4,表5和表6所示，待测抗体可不同程度地结合人和猴TIM-3胞外区蛋白，不能结合鼠TIM-3蛋白。其中IgG对照为大鼠IgG，表中的数据为A450nm数值。
表4酶联免疫吸附实验中TIM-3抗体与人TIM-3胞外区蛋白反应活性
Figure PCTCN2019122471-appb-000009
表5酶联免疫吸附实验中TIM-3抗体与猴TIM-3胞外区蛋白反应活性
Figure PCTCN2019122471-appb-000010
表6酶联免疫吸附实验中TIM-3抗体与鼠TIM-3胞外区蛋白反应活性
Figure PCTCN2019122471-appb-000011
(二)流式细胞实验(FACS)检测抗体与TIM-3表达细胞的结合
将实施例1所述含有编码人源TIM-3全长核苷酸序列的pIRES质粒转染CHOK1细胞株得含人TIM-3的CHOK1稳转细胞株(此处称为CHOK1-hTIM-3稳定细胞株)，将带有猴源TIM-3全长基因的pIRES质粒，转染CHOK1细胞株构建含猴TIM-3的CHOK1稳转细胞株(此处称为CHOK1-cTIM-3稳定细胞株)。将CHOK1-hTIM-3稳定细胞株和CHOK1-cTIM-3稳定细胞株在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS(Hanks'Balanced Salt Solution)洗涤1-2次，然后用无酶细胞解离液(Versene solution:Life technology)处理和收集细胞。用HBSS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用HBSS稀释至1-2x10 6细胞每毫升，加入1％山羊血清封闭液，冰上孵育20-30分钟，然后用HBSS离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA)悬浮至2x10 6细胞/ml,按每孔100微升加入到96孔FACS反应板中，加入待测抗体样品每孔100微升，冰上孵育1-2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗，冰上孵育0.5-1.0小时。用FACS缓冲液离心洗涤2-3次，加入每孔100微升固定液(4％Paraformaldehyde)悬浮细胞，5-10分钟后用FACS缓冲液离心洗涤1-2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACSCalibur,BD)检测和分析结果。结果如图7和图8，表7和表8所示，其中IgG对照为大鼠IgG，表中的数据为MFI所测细胞群的平均荧光强度值。结果表明：待测抗体可结合细胞表面的人或猴TIM-3蛋白。
表7 FACS检测TIM-3抗体与CHOK1-hTIM-3的结合反应
Figure PCTCN2019122471-appb-000012
Figure PCTCN2019122471-appb-000013
表8 FACS检测TIM-3抗体与CHOK1-cTIM-3的结合反应
Figure PCTCN2019122471-appb-000014
(三)淋巴细胞刺激实验检测TIM-3抗体对淋巴细胞活性的影响
淋巴细胞刺激实验检测TIM-3抗体阻断TIM-3蛋白与其受体的结合从而解除其对T淋巴细胞活性的抑制，从而刺激T细胞的增殖。
1.Ficoll分离全血获取外周血单核淋巴细胞PBMC。
将新鲜获取的全血用磷酸缓冲液PBS以1:1的体积比例稀释得稀释后的全血，用无菌吸管轻轻将稀释后的全血铺平在Ficoll液面(购自GE Healthcare)，Ficoll与稀释后的全血的体积比为3:4，避免震荡混匀，以400g转速室温20℃梯度离心30分钟，离心后的离心管分为三层，上层为血浆，中间乳白色分层即为单核淋巴细胞。用无菌吸管轻轻吸取中间层细胞，收集至新的离心管，用PBS磷酸缓冲液稀释至三倍体积，100g转速室温离心10分钟，弃上清。将淋巴细胞用PBS磷酸缓冲液重悬至10mL，重复前面步骤取出血小板。最后将淋巴细胞重悬至10mL含有10％胎牛血清的多组份RPMI1640培养基(购自Invitrogen)备用，即为外周血单核淋巴细胞PBMC，所述百分比为质量百分比。
2.OKT3介导PBMC细胞的预刺激实验
商业化的OKT3抗体(eBioscience Cat#16-0037-81)用PBS稀释到终浓度1.0μg/ml，然后以2ml每孔加到6孔细胞培养板。用塑料膜封好4℃孵育过夜，第二天用PBS洗3次，将分离得到的PBMC细胞接种到6孔板中，于37℃、5％CO 2 培养箱培养72小时。
3.丝裂霉素处理CHOK1-OS8细胞
将抗人类CD3单抗OKT3的单链可变片段(scFv)融合至小鼠CD8a C-末端域(113-220)建构Τ细胞接合物，膜错定嵌合抗体(OS8)，该小鼠CD8a的C末端域包括较链、跨膜及胞质域，这样可以将抗CD3scFv片段错定至细胞表面作为T细胞激活物。将表达重组融合蛋白OS8的质粒转染CHOK1细胞株得到在细胞表面表达OS8(一种T细胞激活分子)的CHOK1稳转细胞株(此处称为CHOK1-OS8稳定细胞株)。使用前，将CHOK1-OS8细胞用10μg/mL丝裂霉素，37℃处2小时，用PBS洗3次以去除残留的丝裂霉素。
4.CHOK1-OS8介导的PBMC刺激实验
试验前，配制等体积比稀释的待测的TIM-3抗体，得待测样品溶液。
将预刺激的外周血单核淋巴细胞PBMC以1×10 5个细胞100微升每孔铺至96孔细胞培养板，然后将所述的待测样品溶液加入培养板，室温培养30分钟。最后加入CHOK1-OS8细胞，以2.5×10 4个细胞50微升每孔铺至96孔细胞培养板，保证每个反应孔200μL体积，将反应板于37℃、5％CO 2培养箱培养20小时后收集上清，得细胞上清液，于-20℃冻存，所述百分比为体积百分比。
5.细胞上清中细胞因子白介素IFN酶联免疫吸附检测。
细胞上清中细胞因子干扰素IFN-γ酶联免疫吸附检测使用R&D system相关检测试剂盒human IFN-γDuoSet ELISA(DY285)，并按照说明书操作。除检测抗体外的所有检测试剂均由检测试剂盒提供。
测定细胞上清中细胞因子IFN-γ含量的酶联免疫吸附检测采用双抗夹心ELISA试剂盒(购自R&D Systems，IFN-γCat#DY285)。实验操作严格按照试剂盒说明书要求，所有检测试剂均由试剂盒提供。具体实验简述如下：将IFN-γ多克隆抗体包被于ELISA微孔板上，用塑料膜封好4℃孵育过夜，第二天用洗板液洗板4次，加入封闭液室温封闭1-2小时。用洗板液洗板4次，将步骤4获得的细胞上清液作为待测样品，加入标准品和待测样品室温孵育2小时。每孔加入400微升洗液，重复洗板4次；再加入抗人IFN-γ的辣根过氧化物酶标抗体，室温孵育2小时，与微孔板上的IFN-γ形成免疫复合物，清洗微孔；加入底物显色，避光室温30分钟，最终加入终止液，用酶标仪测定A450nm吸光度。
检测TIM-3抗体在所述PBMC刺激实验中对IFN-γ分泌的影响。结果如图9，和表9所示。其中IgG对照为大鼠IgG，表中的数据为IFN-γ值(pg/mL)。
表9 TIM-3抗体在OKT3依赖的PBMC激活试验中对IFN-γ分泌的影响
Figure PCTCN2019122471-appb-000015
Figure PCTCN2019122471-appb-000016
结果表明：在PBMC淋巴细胞刺激试验中待测抗体可使PBMC的IFN-γ分泌增强。
实施例3轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例2的检定和活性测定后)，通过离心搜集5×10 7个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％乙醇(所述百分比为体积百分比)，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促溶10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μl PCR体系，包括1μl cDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs。设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒为Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2019122471-appb-000017
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时得连接产物，其中连接的试剂盒为T4 DNA连接酶，购自NEB，货号M0402；取5μl连接产物加入100μl的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟。而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μl PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序。其中，测序的步骤参见Kabat，Sequences of Proteins of Immunological  Interest，National Institutes of Health,Bethesda,Md.(1991)。
测序结果见附录中本发明的序列信息，7个克隆的重链CDR1-3和轻链CDR1-3的序列信息见表18。
实施例4：全人抗体IgG转化和制备
(一)质粒构建与准备：实施例2已从杂交瘤细胞的培养上清液中获得了纯化的TIM-3抗体，并根据实施例3的测序结果明确了TIM-3抗体的重链可变区和轻链可变区序列。将TIM-3抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG4恒定区的表达载体(其中表达载体购买自Invitrogen)中，将TIM-3抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体当中，得重组质粒并经测序验证(测序方法与实施例3中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
(二)细胞转染：
在培养基Freestyle 293表达培养基(expression medium)(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM，8％CO 2(v/v)浓度。
Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。
取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293 expression medium中至293E的细胞密度为1.5×10 6/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
(三)抗体纯化：对于连续生产的无内毒素的层析柱和Protein A填料(购自GE)，使用0.1M NaOH处理30分钟或者5个柱体积的0.5M NaOH冲洗。对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％(v/v)Triton×100对柱料清洗。使用5个柱体积的PBS(PBS磷酸缓冲液，pH7.2)进行平衡，将步骤(2)所得过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用0.5倍柱体积洗脱液的pH8.5的1M Tris-HCl(1.5M NaCl)中和，收获全人TIM-3抗体。上述所用 溶液均需要新配置。收获全人TIM-3抗体后，在1×PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。
实施例5全人TIM-3抗体的鉴定
(一)酶联免疫吸附实验(ELISA)检测抗体与TIM-3蛋白的结合
结果如图10，图11和图12，表10，表11和表12所示。其中IgG对照为人IgG，表中的数据为A450nm数值。
表10酶联免疫吸附实验中全人TIM-3抗体与人TIM-3胞外区蛋白反应活性
Figure PCTCN2019122471-appb-000018
表11酶联免疫吸附实验中全人TIM-3抗体与猴TIM-3胞外区蛋白反应活性
Figure PCTCN2019122471-appb-000019
表12酶联免疫吸附实验中全人TIM-3抗体与鼠TIM-3胞外区蛋白反应活性
Figure PCTCN2019122471-appb-000020
Figure PCTCN2019122471-appb-000021
结果表明：全人TIM-3抗体可结合人和猴TIM-3胞外区蛋白，不能结合鼠TIM-3蛋白。各抗体活性相当，表明抗体与TIM-3结合能力较强。
(二)流式细胞实验(FACS)检测抗体与TIM-3表达细胞的结合
结果如图13和图14，表13和表14所示。其中IgG对照为人IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
表13 FACS检测全人TIM-3抗体与CHOK1-hTIM-3的结合反应
Figure PCTCN2019122471-appb-000022
表14 FACS检测全人TIM-3抗体与CHOK1-cTIM-3的结合反应
Figure PCTCN2019122471-appb-000023
Figure PCTCN2019122471-appb-000024
结果表明：全人TIM-3抗体可不同程度地结合细胞表面的人和猴TIM-3蛋白。
(三)TIM-3受体配体结合实验检测TIM-3抗体阻断TIM-3与其配体磷脂酰丝氨酸的结合
用1μM星形孢菌素在37℃处理Jurkat细胞2小时以诱导细胞凋亡。用Annexin V-FITC细胞凋亡检测试剂盒检测磷脂酰丝氨酸的存在。hTIM-3-hFc与星形孢菌素处理的Jurkat细胞强烈结合，但不与未处理的Jurkat细胞结合。用PBS缓冲液洗涤细胞1-2次,进行细胞计数后将细胞用结合缓冲液稀释至1-2×10 6细胞每毫升，按每孔100微升加入到96孔FACS反应板中。用结合缓冲液配置待测抗体样品与2μg/ml的hTIM-3-hFc蛋白，并等体积混合。将混合物在室温孵育30分钟后，把孵育凋亡细胞的FACS反应板离心弃掉上清，按照每孔100微升将上述的混合物加入凋亡细胞中，室温孵育1小时。用结合缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗，孵育0.5小时。用结合缓冲液离心洗涤2-3次，加入每孔100微升PBS悬浮细胞，用FACS(FACS Calibur,BD)检测和分析结果。结果如表15和图15所示，其中IgG对照为人源IgG，表中的数据为抑制率(％)。
表15全人TIM-3抗体阻断TIM-3蛋白与其受体磷脂酰丝氨酸的结合
Figure PCTCN2019122471-appb-000025
结果表明，所得抗体可不同程度的抑制TIM-3蛋白与其受体磷脂酰丝氨酸的结合，所测抗体活性相当。
(四)淋巴细胞刺激实验检测TIM-3抗体对淋巴细胞活性的影响
淋巴细胞刺激实验检测TIM-3抗体阻断TIM-3蛋白与其受体的结合从而解除其对T淋巴细胞活性的抑制，从而刺激T细胞的增殖。
结果如图16和图17，表16和表17所示，本试验选用2位捐赠者的PBMC细胞，结果基本保持一致。其中hIgG对照为人IgG，表中的数据为IFN-γ值(pg/mL)。
表16全人TIM-3抗体在淋巴细胞激活试验中对IFN-γ分泌的影响(捐赠人X)
Figure PCTCN2019122471-appb-000026
表17全人TIM-3抗体在淋巴细胞激活试验中对IFN-γ分泌的影响(捐赠人Y)
Figure PCTCN2019122471-appb-000027
结果表明：在PBMC淋巴细胞刺激试验中待测抗体可不同程度地使PBMC的IFN-γ分泌增强。
(五)抗体亲和力检测试验
首先，选用AHC传感器，并且用缓冲液平衡传感器10min，然后用缓冲液将待测抗体稀释至5μg/ml，用传感器固化抗体3-5min，信号值高度1-2nm。再用缓冲液平衡传感器3min，接着将抗原蛋白稀释至100nM(最高浓度暂定100nM)，结合和解离传感器偶联的抗体。若得到足够的信号值，然后将抗原蛋白倍比稀释几个浓度梯度，分析抗体抗原的结合和解离。在每个循环结束后，传感器表面用10mM，PH 1.5的Glycine进行再生。动力学速率常数需减去空白对照，用 global fit分析方法1：1结合模型进行数据拟合。解离平衡速率常数(KD)用以下公式计算：KD＝kd/ka。结果如表18所示。
表18抗TIM-3抗体亲和力的分析测定
Clone ID ka(1/Ms) kd(1/s) KD(M)
7A4F10 1.11E+06 1.13E-03 1.01E-09
18D2H2 1.22E+06 3.13E-03 2.58E-09
34B6D8 1.15E+06 1.20E-03 1.05E-09
39E5H1 1.06E+06 1.55E-03 1.46E-09
57F4E5 1.60E+06 3.97E-03 2.48E-09
134H3G6 1.19E+06 4.83E-03 4.05E-09
215A8F2 9.65E+05 1.28E-04 1.33E-10
结果表明：所得全人TIM-3抗体的KD值均在纳摩(nM)水平，表明这些抗体对人TIM-3ECD均有较好的亲和力，其中，215A8F2抗体对人TIM-3ECD的亲和力最好。
讨论
全人抗体
单克隆抗体的应用是过去20年时间肿瘤治疗中最成功及最具变革意义的治疗手段之一。与传统化学药物相比，抗体药物具有更高的特异性及更低的毒性。虽然单抗药物取得了持续不断的成功，但仍面临诸多挑战。
鼠源单克隆抗体最大的缺陷则是它所诱发的HAMA(人抗鼠抗体)反应。因而，鼠单抗在肿瘤、器官移植等疾病的诊断和治疗上有较大的局限性；嵌合抗体仍保留着30％的鼠源序列，可引起不同程度的HAMA反应。临床显示不同的嵌合抗体有着不同程度的免疫原性；人源化抗体又称移植抗体。简单CDR移植常常导致抗原抗体亲和力下降，由于其仍至少具有10％的异源蛋白，在临床应用中还是受到不同程度的限制。因而有待于进一步研制更完善的治疗性抗体—完全人抗体。
1994年，美国Abgenix和Genpham两公司报告了利用转基因小鼠制备完全人抗体，从而解决了人体不能被随意免疫这一人抗体制备研究的难题。此后完全人抗体制备技术的不断发展成熟，所获人单克隆抗体已具有较强的抗肿瘤活性。H2L2转基因鼠是由和铂医药(上海)有限责任公司许可提供，鹿特丹大学医学中心(荷兰，鹿特丹)的Frank Grosveld教授实验室开发的转基因小鼠技术，产生具有完全人类可变区的由两条重链和两条轻链(H2L2)组成的传统四聚体抗体的小鼠。产生的抗体亲和力成熟，可变区完全人源化，具有优异的溶解性。基因工程小鼠技术是产生完全人类抗体的主要工具之一。
免疫治疗
癌症免疫治疗是指通过免疫系统达到对抗癌症目的的治疗方式。近来癌症免疫治疗备受关注，除了手术、化疗及放疗外，已成为癌症治疗的新手段。免 疫检查点是指免疫系统中存在的一些抑制性信号通路，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受。利用免疫检查点的抑制性信号通路抑制T细胞活性是肿瘤逃避免疫杀伤的重要机制。针对免疫检查点的阻断是众多激活抗肿瘤免疫的有效策略之一。
免疫检查点蛋白的抑制剂具有治疗各种肿瘤类型(如转移性黑素瘤，肺癌，乳腺癌，肾细胞癌等)的潜力。最近癌症免疫治疗方法的研究已经显示出可喜的成果，特别是对转移癌癌症病例。此外，癌症免疫治疗在治疗血液癌症方面具有巨大的潜力，包括霍奇金淋巴瘤，多发性骨髓瘤，骨髓发育不良综合征，非霍奇金淋巴瘤等。免疫检查点抑制剂引起的副作用是可以忽略的，可逆的和可控的，有效的免疫检查点抑制剂可以显着提高癌症患者的总生存期。免疫检查点抑制剂还可以与靶向治疗或常规放射治疗和化学疗法结合使用，并且这种组合疗法可有效治疗许多类型的癌症,可能是治疗或者治愈多种癌症的希望。
目前TIM-3抗体的临床研究主要用于恶性实体瘤和淋巴瘤的治疗，而且也主要集中于它和其他疗法或靶点药物的联合使用，开发适应症广的抗体从而扩大其适用的临床症状，包括不可切除的转移性黑色素瘤、晚期实体癌、乳腺癌、子宫内膜癌、卵巢癌、肾癌、胰腺癌、复发性胶质母细胞瘤、头颈癌、膀胱癌、转移性直结肠癌、胃肠道间质肿瘤、腺泡细胞癌、高级恶性固体肿瘤、非小细胞肺癌等。
本发明技术方案的优势：
治疗用单克隆抗体可由多种技术和途径进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等。但是通过杂交瘤技术从野生型或转基因小鼠制备单抗，仍然是目前治疗用单抗制备方法的主流。根据目前最新的单抗技术进展，本发明采用优化的杂交瘤技术来制备所需的抗TIM-3抗体。
①可以通过对野生型小鼠进行免疫获得抗体，但是需要对鼠源抗体进行人源化改造而获得人源化抗体，缺点在于改造后的抗体免疫原性可能更强、抗体结构可能会改变从而导致活性丢失或可生产性变差。
②可以通过对全人源转基因小鼠进行免疫获得全人抗体，但是所获得抗体的数量或亲和力会较差。
③可通过构建免疫小鼠抗体库用噬菌体展示技术对抗体进行表达和活性筛选，但是涉及到抗体重轻链的随机重新组合，会导致形成的抗体可生产性较差。
④可通过构建人源抗体库用噬菌体展示技术对抗体进行表达和活性筛选，但是因为未经免疫，所得抗体亲和力会较差。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明的序列信息(包括本发明TIM-3抗体的CDR区氨基酸序列及其序列编号，本发明TIM-3抗体的重/轻链可变区的氨基酸和基因序列及其序列编号)见下表18
表18
Figure PCTCN2019122471-appb-000028
Figure PCTCN2019122471-appb-000029
Figure PCTCN2019122471-appb-000030
Figure PCTCN2019122471-appb-000031
注：
1.其中VH-CDR1为重链可变区-CDR1，VH-CDR2为重链可变区-CDR2，VH-CDR3为重链可变区-CDR3；其中VL-CDR1为轻链可变区-CDR1，VL-CDR2为轻链可变区-CDR2，VL-CDR3为轻链可变区-CDR3。
2.VH-aa是指重链可变区氨基酸序列，VH-nt是指重链可变区基因序列；VL-aa是指轻链可变区氨基酸序列，VL-nt是指重链可变区基因序列。

Claims (17)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.10n+3所示的VH-CDR1，
    SEQ ID NO.10n+4所示的VH-CDR2，和
    SEQ ID NO.10n+5所示的VH-CDR3；
    其中，各n独立地为0、1、2、3、4、5或6；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.10n+8所示的VL-CDR1，
    SEQ ID NO.10n+9所示的VL-CDR2，和
    SEQ ID NO.10n+10所示的VL-CDR3；
    其中，各n独立地为0、1、2、3、4、5或6；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链，
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
  6. 如权利要求5所述的抗体，其特征在于，所述的抗体具有如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区；
    其中，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
    VH-CDR1 VH-CDR2 VH-CDR3 VL-CDR1 VL-CDR2 VL-CDR3
    序列编号 序列编号 序列编号 序列编号 序列编号 序列编号 3 4 5 8 9 10 13 14 15 18 19 20 23 24 25 28 29 30 33 34 35 38 39 40 43 44 45 48 49 50 53 54 55 58 59 60 63 64 65 68 69 70
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TIM-3结合亲和力的衍生序列。
  7. 如权利要求5所述的抗体，其特征在于，所述抗体的重链可变区含有SEQ ID NO.1、11、21、31、41、51或61所示的氨基酸序列；和/或所述抗体的轻链可变区含有SEQ ID NO.6、16、26、36、46、56或66所示的氨基酸序列。
  8. 如权利要求6所述的抗体，其特征在于，所述的抗体选自下组：
    抗体编号 克隆 VH序列编号 VL序列编号 1 7A4F10 1 6 2 18D2H2 11 16 3 134H3G6 21 26 4 215A8F2 31 36 5 34B6D8 41 46 6 39E5H1 51 56 7 57F4E5 61 66。
  9. 一种重组蛋白，其特征在于，所述的重组蛋白包括：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  10. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体；以及
    (2)如权利要求9所述的重组蛋白。
  11. 如权利要求10所述的多核苷酸，其特征在于，编码所述重链可变区的多核苷酸如SEQ ID NO.2、12、22、32、42、52或62所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO.7、17、27、37、47、57或67所示。
  12. 如权利要求11所述的多核苷酸，其特征在于，编码所述重链可变区序列的多核苷酸和编码所述轻链可变区序列的多核苷酸选自下组：
    Figure PCTCN2019122471-appb-100001
  13. 一种载体，其特征在于，所述载体含有本发明权利要求10-12中任一项所述的多核苷酸。
  14. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有权利要求13所述的载体或基因组中整合有权利要求10-12中任一项所述的多核苷酸。
  15. 一种抗体偶联物，其特征在于，该抗体偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  16. 一种免疫细胞，其特征在于，所述免疫细胞表达或在细胞膜外暴露有权利要求5-8中任一项所述的抗体。
  17. 一种药物组合物，其特征在于，所述药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5-8中任一项所述的抗体、如权利要求9所述的重组蛋白、如权利要求15所述的抗体偶联物、权利要求16所述的免疫细胞、或其组合；以及
    (ii)药学上可接受的载体。
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CN115561458B (zh) * 2022-07-08 2023-12-05 华中科技大学同济医学院附属协和医院 用于诊断胆道闭锁的胶体金快速检测卡及其制备方法

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