WO2019174637A1 - Molécule d'anticorps complètement humanisé contre tim-3, fragment de liaison à l'antigène et utilisation médicale associée - Google Patents

Molécule d'anticorps complètement humanisé contre tim-3, fragment de liaison à l'antigène et utilisation médicale associée Download PDF

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WO2019174637A1
WO2019174637A1 PCT/CN2019/078313 CN2019078313W WO2019174637A1 WO 2019174637 A1 WO2019174637 A1 WO 2019174637A1 CN 2019078313 W CN2019078313 W CN 2019078313W WO 2019174637 A1 WO2019174637 A1 WO 2019174637A1
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amino acid
antibody
acid sequence
binding fragment
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刘佳建
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珠海市丽珠单抗生物技术有限公司
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • Tumor immunotherapy is a major breakthrough in the field of cancer treatment in recent years.
  • Anti-drugs against the immune checkpoint PD-1/PD-L1 Since the beginning of 2014, five drugs have been approved for marketing, but have not yet been listed in China. The total anti-drug sales of PD-1/PD-L1 is close to the $10 billion mark in 2017.
  • PD-1/PD-L1 antibody drugs are only effective in some patients, and a large number of patients have no way to benefit from this huge advancement in tumor immunotherapy.
  • T cells are the center of immunotherapy, and whether T cells can effectively regulate T cells is one of the key factors for the effectiveness of immunotherapy.
  • the PD-1/PD-L1 antibody drug is one of the immunological checkpoint antibody drugs against T cells.
  • the idea of combining immune checkpoint antibodies is to expect better use of T cells as a central role in tumor immunotherapy and is one of the ways to improve the efficiency of cancer patients.
  • the combination of anti-PD-1 antibody Opdivo and anti-CTLA-4 antibodies has been approved for the treatment of melanoma.
  • Other immunological checkpoint antibodies and PD-1/PD-L1 antibody combinations are also considered to be effective ways to increase the effectiveness of PD-1/PD-L1 antibodies, and some are in clinical trials.
  • Recently (2017 ASCO Annual Meeting) new immunological checkpoint antibody combination therapy has been used to improve the dissemination of effective clinical data.
  • the current clinical trial of the TIM-3 antibody is Tesaro's TSR-022 for the treatment of advanced or metastatic solid tumors and Novartis MGB-453, either alone or in combination with PD-1, for use alone or in combination with PD-1 antibodies. Treatment of advanced malignant tumors. There is no more clinical treatment information for antibodies to the TIM-3 target.
  • the present invention obtains an antibody against TIM-3 by a large number of optimized innovation screens.
  • the present invention provides murine antibodies, humanized, fully humanized antibodies, and the like that bind human Tim-3. They have better binding activity, fast binding, slow dissociation, and better activity on human blood cells to activate human blood cells to kill tumor cells, either alone or in combination with PD-1 antibody to increase PD-1 antibody activation. Cellular activity. And the unique site of the human Tim-3 protein should be related to its function. These characteristics make the antibody of the present invention more suitable for drug development, have small immunogenicity, and have long-lasting binding target.
  • the PD-1 antibody alone or in combination can effectively exert the killing effect of human blood cells, including NK cells on tumor cells, to achieve better treatment of tumors. The patient's therapeutic effect and improved PD-1 treatment efficiency.
  • the present invention provides an antibody molecule or a binding fragment thereof which specifically binds to a human T cell immunoglobulin domain and a mucin domain 3 (TIM-3), comprising at least one CDR region sequence selected from the group consisting of Mutation sequence:
  • VL light chain variable region
  • VH heavy chain variable region
  • VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 14 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;
  • VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5, the VLCDR2 amino acid sequence of SEQ ID NO: 6 and the VLCDR3 amino acid sequence of SEQ ID NO: 7; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 9 and the VHCDR3 amino acid sequence of SEQ ID NO: 10;
  • VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15, the VLCDR2 amino acid sequence of SEQ ID NO: 16 and the VLCDR3 amino acid sequence of SEQ ID NO: 17; and VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: The VHCDR2 amino acid sequence of SEQ ID NO: 19 and the VHCDR3 amino acid sequence of SEQ ID NO: 20.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR3 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody light chain variable region comprises the VLCDR3 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR3 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR1 as set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR1 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR2 set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof, as described above, wherein the antibody heavy chain variable region comprises the VHCDR3 as set forth in SEQ ID NO: Amino acid or its mutated sequence.
  • the TIM-3 antibody or binding fragment thereof comprising a light chain variable region (VL) comprising the VLCDR1 amino acid of SEQ ID NO: 5, is provided.
  • VL light chain variable region
  • the TIM-3 antibody or binding fragment thereof comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15 or a variant thereof, SEQ ID NO: The VLCDR2 amino acid sequence of 16 or a variant thereof and the VLCDR3 amino acid sequence of SEQ ID NO: 17 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising a heavy chain variable region (VH) comprising a SEQ ID NO: 8
  • VH heavy chain variable region
  • the TIM-3 antibody or binding fragment thereof comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 11 or a variant thereof
  • VHCDR2 amino acid sequence of SEQ ID NO: 12 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 13 or a variant thereof
  • VHCDR2 amino acid sequence of SEQ ID NO: 14 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 11 or a variant thereof
  • VHCDR2 amino acid sequence of SEQ ID NO: 9 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VH comprising a VHCDR1 amino acid sequence selected from the group consisting of SEQ ID NO: 18 or a variant thereof
  • VHCDR2 amino acid sequence of SEQ ID NO: 19 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 20 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 8 or a variant thereof, SEQ ID The VHCDR2 amino acid sequence of NO: 9 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 11 or a variant thereof, SEQ ID NO: The VHCDR2 amino acid sequence of 12 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 13 or a variant thereof, SEQ ID NO: The VHCDR2 amino acid sequence of 14 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 5 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 6 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 7 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 11 or a variant thereof, SEQ ID The VHCDR2 amino acid sequence of NO: 9 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • the TIM-3 antibody or binding fragment thereof comprising VL comprising the VLCDR1 amino acid sequence of SEQ ID NO: 15 or a variant thereof, SEQ ID NO: a VLCDR2 amino acid sequence of 16 or a variant thereof and a VLCDR3 amino acid sequence of SEQ ID NO: 17 or a variant thereof; and VH comprising a VHCDR1 amino acid sequence selected from SEQ ID NO: 18 or a variant thereof, SEQ ID NO: The VHCDR2 amino acid sequence of 19 or a variant thereof and the VHCDR3 amino acid sequence of SEQ ID NO: 20 or a variant thereof.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or binding fragment thereof is a murine antibody molecule or a binding fragment thereof.
  • a TIM-3 antibody or a binding fragment thereof wherein the TIM-3 antibody or binding fragment thereof is a murine antibody molecule or a binding fragment thereof,
  • the murine TIM-3 antibody molecule or a binding fragment thereof is increased in affinity by affinity by a factor of 3-10 times, preferably 10 times.
  • a TIM-3 antibody or a binding fragment thereof as described above wherein the mouse antibody molecule or binding fragment thereof has a light chain variable region amino acid sequence of SEQ ID NO: 1 or its variant sequence.
  • a TIM-3 antibody or a binding fragment thereof as described above wherein the human antibody molecule or binding fragment thereof has a heavy chain variable region amino acid sequence of SEQ ID NO: 2 or its variant sequence.
  • a TIM-3 antibody or a binding fragment thereof wherein the mouse antibody light chain variable region amino acid sequence is SEQ ID NO: 1 or a variant thereof And the heavy chain variable region amino acid sequence is SEQ ID NO: 2 or a variant thereof.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or binding fragment thereof is a variable of a murine antibody molecule or a binding fragment thereof
  • the region and the human antibody constant region include a chimeric antibody molecule or a binding fragment thereof comprising a human antibody heavy chain constant region IgG1, IgG2, IgG4, IgG4 and a light chain constant region kappa, a lambda chain or the like.
  • a TIM-3 antibody or a binding fragment thereof wherein the chimeric antibody molecule or binding fragment thereof has the light chain amino acid sequence of SEQ ID NO: 25 or Variation sequence.
  • the TIM-3 antibody or binding fragment thereof wherein the chimeric antibody molecule or binding fragment thereof has the heavy chain amino acid sequence of SEQ ID NO: 26 or Variation sequence.
  • a TIM-3 antibody or a binding fragment thereof wherein the chimeric antibody molecule or binding fragment thereof has the light chain amino acid sequence of SEQ ID NO: 25 or A variant sequence, and the heavy chain amino acid sequence is SEQ ID NO: 26 or a variant thereof.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof is a chimeric antibody, and the TIM-3 is chimeric
  • the antibody or its binding fragment is increased in affinity by affinity by a factor of 3-10 times, preferably 10 times.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or binding fragment thereof is a humanized antibody molecule or a binding fragment thereof.
  • a TIM-3 antibody or a binding fragment thereof, wherein the humanized antibody molecule or binding fragment thereof, the light chain variable region framework FR sequence is selected from the group consisting of Germline light chain sequence, including IGKV1D-39*01(F), IGKV1-12*01(F), IGKV1-39*01(F), IGKV1-12*02(F), IGKV1-17*01(F ), IGKV1-27*01(F), IGKV1-39*02(P), IGKV1-6*01(F), IGKV1-NL1*01(F), IGKV1D-12*01(F), etc., preferably the same
  • the source, and the preferred frequency of the germline, etc., preferably IGKV1-39*01(F) is a germline light chain for humanization.
  • the light chain J gene hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, hJK3 and the like have high homology, preferably hJK2.1.
  • the FR sequence region preferably has a 0-10 amino acid back mutation.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein said humanized antibody molecule or binding fragment thereof has a light chain variable region CDR sequence according to Kabat, respectively
  • Each CDR sequence as defined by the numbering rules of Chothia, AbM, Contact, or CCG, the CDR sequences comprising the light chain CDR sequences set forth in Tables 3-6, Table 2b, or variants thereof.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof has a light chain variable region sequence selected from the group consisting of SEQ ID NO: 27-33 or its variant sequence.
  • a TIM-3 antibody or a binding fragment thereof wherein the humanized antibody molecule or binding fragment thereof, the heavy chain variable region framework FR sequence is selected from the group consisting of Germline heavy chain sequence, including IGHV3-7*01(F), IGHV3-7*02(F), IGHV3-7*03(F), IGHV3-48*01(F), IGHV3-48*02(F ), IGHV3-48*03 (F), IGHV3-21*01 (F), IGHV3-21*02 (F), IGHV3-21*03 (F), etc., preferably IGHV3-21*01 (F).
  • the FR sequence region preferably has a 0-10 amino acid back mutation.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein said humanized antibody molecule or binding fragment thereof has a heavy chain variable region CDR sequence according to Kabat, respectively
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof has a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 34-37 or its variant sequence.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody comprises a sequence selected from the group consisting of SEQ ID NO: 27-33 or variants thereof A light chain variable region and a combination of a heavy chain variable region selected from the group consisting of SEQ ID NO: 34-37 or variants thereof.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof has a light chain comprising a human ⁇ or ⁇ chain constant Zone or its variant.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment heavy chain thereof comprises a human antibody IgG1, IgG2, IgG4, IgG4 heavy chain constant region or variant thereof.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof has a light chain comprising SEQ ID NO: 38, SEQ ID NO: 40, or SEQ ID NO: 43 or a full length light chain sequence having at least 85% sequence homology thereto.
  • the TIM-3 antibody or binding fragment thereof wherein the humanized antibody molecule or binding fragment heavy chain thereof comprises SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 42 or a full length heavy chain sequence having at least 85% sequence homology thereto.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains
  • the humanized antibody light and heavy chain combination SEQ ID NO: 38 and SEQ ID NO: 39.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 40 and SEQ ID NO: 39.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 38 and SEQ ID NO: 41.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 40 and SEQ ID NO: 41.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 38 and SEQ ID NO: 42.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 43 and SEQ ID NO: 42.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein the humanized antibody molecule or binding fragment thereof is selected from humanized light and heavy chains Combination, preferably, the humanized antibody light and heavy chain combination: SEQ ID NO: 43 and SEQ ID NO: 39.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof is a humanized antibody, the TIM-3 human source
  • the affinity of the antibody or its binding fragment is increased by a factor of 3-10 times, preferably 10 times, by affinity.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the anti-TIM-3 antibody or a binding fragment thereof is a murine antibody, a humanized antibody,
  • the humanized antibody, the anti-IM-3 murine antibody, the humanized antibody, the fully humanized antibody antibody or the binding fragment thereof biacore detection shows the characteristics of fast binding and slow dissociation; preferably, the dissociation rate kd is 10 -4 magnitude.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof comprises an antigen-binding fragment of a half antibody or a half antibody, preferably , comprising Fab, F(ab")2, Fv or a single-chain Fv fragment (scFv).
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof binds to a human blood cell PBMC antigen, enhancing PBMC against tumor cells The killing effect.
  • a TIM-3 antibody or binding fragment thereof as described above, wherein said TIM-3 antibody or binding fragment thereof enhances growth of human blood T cells against tumor cell growth Inhibition.
  • a TIM-3 antibody or a binding fragment thereof as described above wherein the TIM-3 antibody or a binding fragment thereof enhances secretion of INF- ⁇ by activated human blood T cells .
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof synergizes with a PD-1 antibody enhances activated human blood T Inhibition of tumor cell growth by cells.
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof synergizes with a PD-1 antibody enhances activated human blood T The cells secrete INF- ⁇ .
  • a TIM-3 antibody or a binding fragment thereof as described above, wherein the TIM-3 antibody or a binding fragment thereof is the same as human Tim-3, macaque (Macaca) Tim-3 Marmoset Tim-3 protein binding activity is different.
  • the present invention further provides an expression vector for expressing a DNA molecule of the above TIM-3 antibody molecule or a binding fragment thereof.
  • a method of treating cancer comprising a TIM-3 antibody as described above, the cancer being preferably lung cancer, melanoma, renal cancer, colorectal cancer of the breast, liver cancer, Metastatic lesions of pancreatic cancer, bladder cancer, leukemia, etc. or cancer.
  • Figure 2b shows the specific binding activity of the anti-TIM-3 humanized, fully humanized antibody and human Tim-3+ cells of the invention.
  • Figure 4a shows the binding activity of the anti-TIM-3 humanized, fully humanized antibody and macaque (Tim-3) protein of the present invention.
  • Figure 4b shows the binding activity of the anti-TIM-3 humanized, fully humanized antibody and marmoset Tim-3 protein of the invention.
  • TIM-3 includes isoforms, mammalian (eg, human) TIM-3, human TIM-3 species homologs, and analogs comprising at least one co-epitope with TIM-3.
  • the amino acid sequence of TIM-3 e.g., human TIM-3 is known in the art, for example, Sabatos et al, 2003. Nat Immunol, 4(11): 1102.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each type of Ig in the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgGl, 2, 3, 4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to VLCDR1, VLCDR2, and VLCDR3; the three CDR regions of the heavy chain refer to VHCDR1, VHCDR2 and VHCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (VLCDR1-3, VHCDR1-3), Chothia, AbM, Contact, and CCG in number and position. Numbering rules (see Table 2, CDR definition methods; Table 2b, CGG definitions).
  • the CDR regions of the light chain and heavy chain of the antibody of the present invention define respective CDR sequences according to the numbering rules of Kabat, Chothia, AbM, or Contact, respectively, as shown in Table 2, Table 2b, and Table 3-6.
  • murine antibody is in the present invention a monoclonal antibody to human TIM-3 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the TIM-3 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine TIM-3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the mouse variable region gene is The human constant region gene is ligated into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic cell, industrial system or prokaryotic system.
  • the antibody light chain variable region of the TIM-3 chimeric antibody further comprises a light chain FR region of a murine kappa, a lambda chain or a variant thereof, an antibody light chain variable region
  • the amino acid sequence is shown in SEQ ID NO: 3.
  • the antibody heavy chain variable region of the TIM-3 chimeric antibody further comprises a heavy chain FR region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the antibody heavy chain variable region amino acid sequence is SEQ ID NO: 4 is shown.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) IgG1.
  • the ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc fragment on IgG.
  • the modification refers to mutations in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.
  • humanized antibody also referred to as CDR-grafted antibody, refers to the CDR sequence of a mouse, in particular, the CDR of the TIM-3 antibody of the present invention is according to Kabat , CDR sequences defined by the numbering rules of Chothia, AbM, Contact or CGG (see Table 2, Table 2b, Tables 3-6), antibodies produced by transplantation into the variable region framework of human antibodies. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr.
  • IMGT ImMunoGeneTics
  • the CDR sequences of the TIM-3 humanized antibody mouse are selected from the group consisting of the light chain SEQ ID NO: 5, 6, 7; the heavy chain SEQ ID NO, 8, 9, 10 .
  • the sequence is selected from the light chain SEQ ID NO: 5, 6, 7; the heavy chain SEQ ID NO, 11, 9, 10.
  • the human antibody variable region framework is designed and selected, wherein the light chain FR region sequence on the antibody light chain variable region is derived from the combined sequence of human germline light chain IGKV1-39*01(F) and hJK2.1 SEQ ID NOs: 27-33, comprising the FR1, FR2, FR3 region of human germline light chain IGKV1-39*01 (F) and the FR4 region of hJK2.1; wherein the heavy chain on the heavy chain variable region of the antibody FR region sequence, derived from the human germline heavy chain IGHV3-21*01 (F) and hJH4.1 combination sequence SEQ ID NO: 34-37, comprising FR1 of human germline heavy chain IGHV3-21*01 (F) , FR2, FR3 area and FR4 area of hJH4.1.
  • the human antibody variable region may be subjected to minimal back mutation, and the site of the back mutation may be 10 or more,
  • the “fully humanized antibody” of the present invention means that the antibody has a human antibody germline sequence other than the CDR sequence derived from the murine antibody.
  • the antibody obtained by the above humanization method has a number of back-mutation sites of light and heavy chains of 0 in the process of humanization, that is, the light-weight chain FR region includes the J region, which is a complete human antibody germline sequence. Also included is that the FR region of the light and heavy chain, including the J region, is the majority of the CDR regions, even 1-6 intact CDRs, preferably one complete CDR sequence and the corresponding CDRs of the human germline antibody, in addition to the entire human antibody germline sequence. The sequence is 100% consistent.
  • Antibody molecules comprise diabodies and single-stranded molecules as well as antigen-binding fragments of antibodies (eg, Fab, F(ab')2 and Fv).
  • the antibody molecule comprises or consists of a heavy chain and a light chain (referred to as a half antibody in the present invention).
  • Fab', F(ab')2, Fc, Fd, Fv single chain antibodies (eg, scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (eg, , humanized) antibodies, which can be produced by modifying intact antibodies, or those that are synthesized de novo using recombinant DNA techniques.
  • Antibodies and antibody fragments can be from any antibody class including, but not limited to, IgG, IgA, IgM, IgD, and IgE and from any antibody subclass (eg, IgGl, IgG2, IgG3, and IgG4). Preparation of antibody molecules can be monoclonal or polyclonal.
  • the antibody may also be a human antibody, a humanized antibody, a CDR-grafted antibody, or an antibody produced in vitro.
  • the antibody may have, for example, a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 or IgG4.
  • the antibody may also have, for example, a light chain selected from kappa or lambda.
  • immunoglobulin (Ig) is used interchangeably with the term "antibody” in the present invention.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Constantly modified or “conservative substitution or substitution” refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein.
  • a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
  • Exogenous refers to a substance that is produced outside of a living organism, cell, or human body depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds of the invention or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high antibody concentrations.
  • a sterile injectable solution can be prepared by combining the active compound (i.e., antibody or antibody portion) in the required amount in combination with one of the ingredients or ingredients listed above, in a suitable solvent, and, if necessary, followed by filtration and disinfection. .
  • compositions, combination therapies of the invention may be combined with other active agents or treatments, the methods comprising administering to the subject an amount of an anti-TIM of the invention effective to treat or prevent a disease (eg, cancer) -3 antibody molecule, optionally in combination with one or more inhibitors of PD-1, PD-L1, PD-L2, LAG-3, CEACAM-1 and/or CEACAM-5 or CTLA-4 antibodies
  • a disease eg, cancer
  • additional active agents or all may be administered in an amount or dose that is higher, lower or equal to each of the individual (eg, as monotherapy) The amount or dose of active agent.
  • a “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • a family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), with uncharged polar side Chain amino acids (eg glycine, aspartame, glutamic acid, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg alanine, arginine) , leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), amino acids with self-branched side chains (eg threonine, tyrosine, isoleucine) An acid), and an amino acid having an aromatic side chain (for example, tyrosine, pheny
  • the term “competition” or “cross-competition” is used interchangeably in the present invention to mean the ability of an antibody molecule to interfere with the binding of an anti-TIM-3 antibody molecule to a target (eg, human TIM-3). Interference with binding can be direct or indirect (eg, by allosteric modulation of the antibody molecule or target).
  • a competitive binding assay eg, FACS assay, ELISA, or BIACORE assay
  • epitopope refers to a portion of an antigen (eg, human TIM-3) that specifically interacts with an antibody molecule.
  • Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions described herein.
  • Antibody molecules directed against TIM-3 can be combined with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and sugar molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines.
  • the combination further includes an inhibitor or activator of an immunological checkpoint modulator (eg, a Tim-3 inhibitor (eg, an anti-TIM-3 antibody molecule), a PD-L1 inhibitor (eg, an anti-PD-L1 antibody molecule), PD -1 inhibitor (eg, anti-PD-1 antibody molecule), or CTLA-4 inhibitor (eg, anti-CTLA-4 antibody), or any combination thereof.
  • a Tim-3 inhibitor eg, an anti-TIM-3 antibody molecule
  • a PD-L1 inhibitor eg, an anti-PD-L1 antibody molecule
  • PD -1 inhibitor eg, anti-PD-1 antibody molecule
  • CTLA-4 inhibitor eg, anti-CTLA-4 antibody
  • TIM-3 blocking may also be combined with standard cancer treatment.
  • TIM- 3 Blocking can be effectively combined with a chemotherapeutic regimen. In these cases, the dose of chemotherapeutic agent administered can be reduced.
  • the composition can be administered in combination with one or more, immunomodulatory agents (eg,
  • anti-TIM-3 antibody molecules include administration of an anti-TIM-3 antibody molecule in combination with a modulator of a co-stimulatory molecule or an inhibitory molecule (eg, a co-inhibitory ligand or receptor).
  • a modulator eg, an agonist of a costimulatory molecule.
  • agonists of co-stimulatory molecules are selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD13 7), GITR, CD30, CD40, An agonist of a BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand (eg, an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion).
  • the anti-TIM-3 antibody molecule is administered in combination with an inhibitor of an immunological checkpoint molecule (or immunosuppressive molecule).
  • Immunological checkpoint refers to a group of molecules on the cell surface of an immune cell that can act as a "gate” to down-regulate or suppress an immune response, such as an anti-tumor immune response.
  • Immunological checkpoint molecules include, but are not limited to, PD-1, PD-L1, cytotoxic T lymphocyte antigen 4 (CTLA-4), B7-H1, B7-H3, OX-40, 4-1BB (CD137), CD40 , TIM-3 and lymphocyte activation gene 3 (LAG-3), and so on.
  • the anti-TIM-3 antibody molecule is administered in combination with an anti-TIM-3 antibody or antigen-binding fragment thereof, and the anti-TIM-3 antibody molecule is administered in combination with an anti-PD-1 antibody or antigen-binding fragment thereof.
  • the anti-TIM-3 antibody molecule is administered in combination with an anti-TIM-3 antibody and an anti-PD-1 antibody or antigen-binding fragment thereof.
  • a bispecific antibody is administered comprising an anti-TIM-3 antibody molecule and an anti-PD-1 or anti-TIM-3 antibody or antigen-binding fragment thereof, or a TIM-3 antibody and a LAG-3 antibody or antigen-binding fragment thereof.
  • kidney cancer such as renal cell carcinoma (RCC)
  • an anti-TIM-3 antibody molecule alone or in combination with another immunomodulatory agent (eg, an anti-LAG-3, anti-PD-1 or anti-PD-L1 antibody molecule)
  • another immunomodulatory agent eg, an anti-LAG-3, anti-PD-1 or anti-PD-L1 antibody molecule
  • CCRCC clear cell renal cell carcinoma
  • metastatic RCC eg, clear cell renal cell carcinoma (CCRCC) or metastatic RCC.
  • Anti-TIM-3 antibody molecules can be administered in combination with one or more of: an immune-based strategy (eg, interleukin 2 or interferon alpha), Targeted drugs (eg, VEGF inhibitors such as monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors such as sulphonic acid, sorafenib, axidini and paclitaxel; RNAi inhibitors)
  • VEGF inhibitors such as monoclonal antibodies against VEGF
  • VEGF tyrosine kinase inhibitors such as sulphonic acid, sorafenib, axidini and paclitaxel
  • RNAi inhibitors an inhibitor of a downstream mediator of VEGF signaling, for example, an inhibitor of a mammalian target of rapamycin (mTOR).
  • mTOR mammalian target of rapamycin
  • Cancer includes but is not limited to basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, primary CNS lymphoma, central nervous system (CNS) tumor, breast cancer, cervical cancer, choriocarcinoma, Colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intradermal tumor, kidney cancer, laryngeal cancer, blood disease (including acute marrow) Leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic or acute leukemia), liver cancer, lung cancer (eg small cell and non-small cell carcinoma), lymphoma, including He Jiejin And non-Hodgkin's lymphoma, lymphocytic lymphoma, melanoma, such as malignant melanoma in the skin or eye, myeloma, neuroblastoma, oral cancer
  • the invention is further described in the following examples, which are not intended to limit the scope of the invention.
  • the experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions, such as the cold spring harbor antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • Reagents without specific source are routine reagents purchased from the market.
  • the antigen used in the present invention was purchased from different companies, TIM-3-his (Cat. No.: TM3-H5229), TIM-3-hFc (Cat. No.: TM3-H5258) was purchased from Beijing Baipusisi Biotechnology Co., Ltd.; TIM -3-his-hFc was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., article number: 10390-H03H; or purified by expression of the present invention.
  • the expressed human Tim-3 protein sequence was UniProtKB/Swiss-Prot: Q8TDQ0.3, the extracellular (ECD) region of HAVR2_HUMAN (amino acids 22-199).
  • 293 cells were expanded in Gibco FreeStyle 293 Expression Medium (Gibco, Cat #12338018) medium. Before the start of the transient, adjust the cell concentration to 6 ⁇ 8 ⁇ 10 5 cell / ml, 1% FBS (Aus Gene X FBS Excellent supplier: AusGeneX, China, Cat # FBSSA500-S), 37 ° C 8% CO2 shaker culture At 24h, the microscopic examination survival rate was >95%, and the cell concentration was 1.2 ⁇ 10 6 cell/ml.
  • Gibco FreeStyle 293 Expression Medium Gibco, Cat #12338018
  • the PEI was slowly added to the plasmid, incubated at room temperature for 10 min, and the plasmid PEI mixed solution was slowly dropped while shaking the culture flask, and the mixture was cultured for 5 days at 37 ° C in an 8% CO 2 shaker, and the supernatant was taken at 3300 G for 10 min for purification.
  • Antibody or -Fc fusion protein purification The sample was centrifuged at high speed to remove impurities, and the gravity column containing Protein A (Mabselect, GE Healthcare Life Science, Cat #71-5020-91AE) was equilibrated with PBS pH 7.4 (Bio-Bio, Cat# F506606-0001), 2-5 column volume rinse. Pass the sample through the column. The column was washed with 5-10 column volumes of PBS (Biotech, Cat# B548117-0500). The target protein was eluted with pH 3.5 0.1 M acetic acid, then adjusted to neutral with Tris-HCl at pH 8.0, and the concentration was determined by a microplate reader, and the mixture was stored and stored for use.
  • PBS pH 7.4 Bio-Bio, Cat# F506606-0001
  • PBS Biotech, Cat# B548117-0500
  • the 293T cell culture medium was changed to 4 ml DMEM high-sugar medium (Shanghai Yuanpei Biotechnology Co., Ltd. / Yuanpei Bio: Cat# L130KJ).
  • CHO-K1 cells Choinese Academy of Sciences, Culture Collection Committee Cell Bank Cat# SCSP-507 were inoculated into a 10 cm culture dish to bring the number of cells to 5 ⁇ 10 5 .
  • 293T cell supernatant (virus) was collected, filtered through a 0.45 ⁇ m filter to cultured CHO-K1 cells, and 10 ug/ml polybrene (Shanghai Shengsheng Biotechnology Co., Ltd.
  • CHO-K1 cells were passaged on day 7, and cells passaged on day 8 were initially screened by adding 10 ug/ml puromycin (Source Culture, Cat# S250J0). After 2-3 days, the cells died in large amounts, and the medium was changed to continue the culture until the cells no longer died. The cells were expanded in a large amount, and the monoclonal cell strains were screened, expanded, and stored frozen.
  • the cell line with high expression of human human Tim-3 obtained in the above example was expanded, and then incubated at 6 ⁇ 10 4 /well 96-well plate, incubating at 37 ° C overnight, and then removing the supernatant, and using immunostaining fixative ( Shanghai Biyuntian Biotechnology Co., Ltd. Cat#P0098) 100 ul / hole fixed at room temperature for half an hour. After washing with PBS (source culture, Cat# B320), 5% milk was blocked at 37 ° C for 2 hours, and PBST was washed 3 times. Add the sample to be tested (human or murine antibody, Jackson Immuno Research). Incubate for 1 hour at 37 ° C and wash 3 times with PBST.
  • immunostaining fixative Shanghai Biyuntian Biotechnology Co., Ltd. Cat#P0098
  • Example 4 Anti-human TIM-3 antibody human blood cell killing (NK) activity against tumor cells
  • the primary screening of the hybridoma cell line includes the detection of the binding activity of the antibody in the secretory supernatant of the hybridoma cell line and the human TIM-3 by the ELISA method of Example 3, and selecting the cloned supernatant having good activity to carry out the method of Example 4
  • the method detects the activity of the secreted antibody in human blood cells, preferably cloned, and the results of partial preferred cloning are shown in Table 1.
  • the initial clones in Table 1 were further subjected to limiting dilution, and after 7-10 days after each dilution, the NK method was used to re-detect the binding and NK activity of the secreted antibody (supernatant) of each clone by the ELISA method described above. After several times of limiting dilution, it was unexpectedly found that the secreted supernatant of the monoclonal cell line 3.13H9G6A6D8 obtained from the actual clone of 3.13H9 maintained a good binding activity and human blood cell NK activity.
  • the antibody sequence is extracted from this monoclonal to obtain the preferred murine mab15 antibody of the present invention.
  • Example 6 Screening and identification of murine anti-human TIM-3 antibody
  • the process of extracting antibody sequences from monoclonal cell lines preferably obtained from hybridomas is a method commonly used by those skilled in the art. Specifically, the above monoclonal cell strain is collected, and after expansion and culture, 1 ⁇ 10 6 cells are taken, and RNA is extracted with Trizol (Invitrogen, 15596-018) (according to the kit instructions), and the extracted RNA is reverse-transcribed into cDNA.
  • the reverse transcription kit was purchased from Biotech Biotechnology (Shanghai) Co., Ltd., Cat# B532435.
  • the cDNA obtained by reverse transcription was used as a template to carry out PCR amplification.
  • the amplified product was sequenced to obtain the light/heavy chain variable region base/coding sequence of the mab15 antibody (see below).
  • the primers used are referred to the manual TB326 Rev. C0308 published by Novagen.
  • amino acid sequence encoded by the base sequence of the light heavy chain variable region of the above monoclonal antibody obtained by the present invention is SEQ ID NO: 3 and SEQ ID NO: 4 below.
  • Monoclonal antibody mab15 light chain variable region protein sequence obtained in a preferred hybridoma monoclonal cell line of the invention:
  • the monoclonal antibody mab15 heavy chain variable region sequence obtained in the preferred hybridoma cell monoclonal strain of the present invention is the monoclonal antibody mab15 heavy chain variable region sequence obtained in the preferred hybridoma cell monoclonal strain of the present invention:
  • the light and heavy chain CDR region sequences of the present invention are determined as follows according to the definition/annotation of the above antibody CDRs.
  • Anti-human TIM-3 antibodies of the invention define CDR sequences according to Kabat
  • Anti-human TIM-3 antibodies of the invention define CDR sequences by AbM
  • Light chain CDR1 RASENIYSYLT (SEQ ID NO: 5)
  • Light chain CDR2 NAKTLAE (SEQ ID NO: 6)
  • Light chain CDR3 QQHYGTPLT (SEQ ID NO: 7)
  • Heavy chain CDR1 GFTFSDYYMT (SEQ ID NO: 11)
  • Heavy chain CDR2 SINYDGRNTY (SEQ ID NO: 12)
  • Heavy chain CDR3 GYYYYGSSPNYFDY SEQ ID NO: 10.
  • Anti-human TIM-3 antibodies of the invention are defined by Chothia CDR sequences
  • Anti-human TIM-3 antibodies of the invention are defined by Contact CDR sequences
  • variable region CDRs of the light and heavy chains of the antibodies of the present invention are as follows, according to the CCG antibody CDR definition method in the art.
  • Anti-human Tim-3 antibodies of the invention define CDR sequences by CCG
  • Light chain CDR2 NAKTLAE (SEQ ID NO: 6) Light chain CDR3 QQHYGTPLT (SEQ ID NO: 7) Heavy chain CDR1 GFTFSDYYMT (SEQ ID NO: 11) Heavy chain CDR2 SINYDGRNTYYLDSLKS (SEQ ID NO: 9) Heavy chain CDR3 GYYYYGSSPNYFDY (SEQ ID NO: 10)
  • the antibody of the present invention can be combined with the above-mentioned light and heavy chain constant regions, and the chimeric antibody can be obtained by the method of expression and purification of Example 1.
  • the chimeric antibody may be a chimeric antibody of human IgG4-kappa chain (hIgG4), and the present invention is represented by the IgG4-k. This chimeric antibody is hereinafter referred to as mab15-c.
  • amino acid sequence of the mab15-c light chain of the present invention is derived from the amino acid sequence of the mab15-c light chain of the present invention:
  • amino acid sequence of the mab15-c heavy chain of the present invention is the amino acid sequence of the mab15-c heavy chain of the present invention:
  • the mab15-c antibody was cloned, expressed and purified as in Example 1.
  • the binding activities of mab15-c and human TIM-3 were examined as described in Example 3, and the results are shown in Table 7 below and Figure 1.
  • control (positive control, control molecule) antibody of the present invention uses ABTIM3, see patent WO2015117002A1
  • the affinity of the antibodies of the invention and human TIM-3 was determined using a Biacore T200, GE Healthcare instrument.
  • Protein A (Thermo Pierce, Cat #21181) was first coupled to the biosensing chip CM5 (Cat.) using pH 7.4 running buffer HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P20).
  • HBS-EP+ 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% P20.
  • #BR-1005-30, GE the chip was activated with freshly prepared 50 mM NHS (N-hydroxysuccinimide) and 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and then injected at pH 4.0 10 mM.
  • the above Biacore data indicates that the antibody of the present invention shows very good binding activity (both ELISA and Biacore are 1/10 nM grade), and surprisingly, its binding to specific antigen shows rapid binding (ka is 10 5 ) and slow solution. Off (kd is 10 -4 ) characteristics. In particular, the kd is 10 - 4 is 10 times slower than the usual 10 -3 .
  • another antibody (mabn) obtained in the screening process of the present invention although its KD (0.78 nM) and the preferred antibody mab15-c KD (0.31 nM) of the present invention are in the same order of 1/10 nM, the dissociation speed is higher than mab15- c should be nearly an order of magnitude (kd is 10 -3 ). That is, the antibody and the human Tim-3 antigen rapidly bind and rapidly dissociate. Such antibodies are thus not preferred antibodies of the invention.
  • These features of the preferred antibody mab15c of the present invention make this antibody molecule a great pharmaceutical advantage for therapeutic antibody drug development.
  • an anti-human TIM-3 antibody mab15 which binds to an unexpectedly preferable activity of the activity is obtained by immunizing a mouse, and the antibody is further humanized in this embodiment. Humanization is carried out as disclosed in many documents in the art.
  • the marker recognizes the CDR regions of the light and heavy chains of the antibody (see Example 6 above for details).
  • the murine antibody sequence was compared with the human antibody germline database (v-base) to find a highly homologous human antibody light chain germline, including IGKV1D-39*01(F), IGKV1-12*01(F) , IGKV1-39*01(F), IGKV1-12*02(F), IGKV1-17*01(F), IGKV1-27*01(F), IGKV1-39*02(P), IGKV1-6* 01(F), IGKV1-NL1*01(F), IGKV1D-12*01(F), etc., preferably homology, and preferred frequency of germline, etc., preferably IGKV1-39*01(F) is a human source The germline light chain is used.
  • the light chain J gene hJK1, hJK2.1, hJK2.2, hJK2.3, hJK2.4, hJK3 and the like have high homology, and the sequence alignment is preferably hJK2.1.
  • the human antibody germline having high homology with the heavy chain of the antibody of the present invention comprises IGHV3-7*01(F), IGHV3-7*02(F), IGHV3-7*03(F), IGHV3-48*01 ( F), IGHV3-48*02 (F), IGHV3-48*03 (F), IGHV3-21*01 (F), IGHV3-21*02 (F), IGHV3-21*03 (F), etc., preferably IGHV3-21*01(F).
  • the CDR region of the antibody mab15 of the present invention was transplanted onto the selected humanized template and recombined with the IgG light and heavy chain constant regions.
  • the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation and the CDR regions are Chemically labile amino acid residues are optimized to provide an optimized anti-TIM-3 humanized series of antibody molecules of the invention.
  • Humanized light chain variable region sequences of the invention are humanized light chain variable region sequences of the invention:
  • Humanized heavy chain variable region sequences of the invention are humanized heavy chain variable region sequences of the invention:
  • the back mutation can be 10 or more, preferably 0-10, such as the sequence of the light chain SEQ ID NO: 27-33. These arbitrary sequences are combined with a human antibody light chain constant region kappa chain or lambda chain, for example, with the light chain constant region sequence set forth in SEQ ID NO: 21 to give one of the light chain sequences of the antibody of the present invention.
  • the heavy chain used for humanization also has a different number of back mutations, and the number of back mutations can be 10 or more, preferably 0-10, as listed in SEQ ID NO: 34-37 of the heavy chain variable region sequence described above. of.
  • heavy chain variable region sequences containing different numbers of back mutations are recombined with the heavy chain constant region sequence selected from the above SEQ ID NOS: 22-24 to obtain one of the heavy chain sequences of the present invention.
  • some light and heavy chain sequences are listed, as shown in the following table.
  • Partially humanized antibody sequence of the invention humanized ab16 antibody amino acid sequence:
  • the recombinant antibody was cloned, expressed and purified by the method of Example 1, and the binding activity of these humanized antibodies and human TIM-3 was detected by the ELISA method described in Example 3, and the binding activity of these antibodies and hTim-3+ cells was examined.
  • Method of Example 2 and these antibodies activate the functional activity of human blood cell killer (NK) tumor cells (Example 4), and the results are shown in Table 11 below and Figure 2a (listing partial antibody and human Tim-3 antigen binding) Graph), Figure 2b (listed partial antibody and human Tim-3+ cell binding profile), and Figure 3 (antibody of the invention activates human blood cell killing tumor cell activity).
  • Antibody of the invention ELISA (nM) Mab15-c 0.183 Ab16 0.145 Ab17 0.212 Ab23 0.210 Ab24 0.243 Ab30 0.233 Ab32 0.184 Ab18 0.198
  • the detection activity of the binding activity of the murine antibody of the present invention and the humanized antibody and human Tim-3+ cells is between 0.112 and 0.161 nM
  • FIG. 2b lists the humanized, fully humanized antibody of the present invention.
  • the results in Figure 3 show that the fully humanized antibody ab32 of the present invention activates human blood cell killing of tumor cell antibody functional activity.
  • 1 ug/ml, 10 ug/ml, 30 ug/ml of the control negative antibody (unrelated human IgG antibody) produced only a background value of 4.7% on average.
  • the positive control antibody and ab32 activity values were subtracted from the background value of 4.7%, and the results are shown in Fig. 3.
  • the results showed that the activity of the positive control antibody to activate human blood cells to kill tumor cells was 1.25%, 4.98%, and 7.9%, respectively, at 1 ug/ml, 10 ug/ml, and 30 ug/ml antibody concentration.
  • the antibody ab32 of the present invention activated human blood cells to kill tumor cells.
  • the activity was 3.08%, 13.1%, and 12.4%, respectively. That is to say, the activity of the antibody ab32 in the present invention was 2.5 times, 2.6 times and 1.6 times, respectively, of the positive control.
  • the activity of ab32-activated human blood cells to kill tumor cells has reached a peak at 10 ug/ml.
  • the activity of the positive antibody was higher than 30 ug/ml.
  • the murine antibody mab15 and human Tim-3 found in the present invention have a good binding activity, and the binding speed is fast and the dissociation is slow. Suitable for the development of therapeutic antibody drugs, including for cancer therapy.
  • the humanized antibody obtained after optimization retains the binding activity of the murine antibody.
  • the humanized antibody obtained by the present invention may have multiple back mutation sites, which are back-mutated into a murine antibody, and retain the same activity characteristics as the humanized antibody and mab15.
  • the humanized antibody (ab32, Table 11) is optimized as a fully humanized antibody, ie, the antibody is derived from the human antibody germline sequence except that the CDR sequence is derived from the murine antibody mab15 sequence, and The antibody retained the binding activity and binding characteristics of mab15, and the binding activity to human Tim-3 was better than that of the positive antibody (Table 7). It retains the characteristics of fast bonding speed and slow dissociation speed. More unexpectedly, the antibody activates human blood cells to kill tumor cell activity (NK kills tumor cell activity) more than twice as much as positive antibodies (10 ug/ml ab32 activity is even better than 30 ug/ml positive control activity). All of these unexpectedly significant advantages make the antibodies of the invention more suitable for the development of monoclonal antibody drugs for tumor therapy against the Tim3 target.
  • Example 9 Activity detection of anti-TIM-3 antibody of the present invention in mixed lymphocyte reaction (MLR assay)
  • the method of detecting INF- ⁇ secretion by a mixed lymphocyte reaction was used to evaluate the activity of the series of antibodies of the present invention on activating human blood cells. That is, the human blood cell PBMC isolated from the present invention (isolated from the peripheral blood of a healthy volunteer) was induced to obtain dendritic cells (DC), and T cells from different volunteers were stimulated.
  • MLR assay mixed lymphocyte reaction
  • DC dendritic cells
  • TNF- ⁇ was purchased from Peprotech, Cat#: AF-300-01A), and culture was continued for 2 days to obtain dendrites.
  • DC Cells
  • DC stimulation of PBMC/T cells (MLR) assay 10 ng/ml anti-CD3 antibody (Miltenyl Biotec, Cat#: 130-093-387), coated with 96-well cell culture plates at 100 ul/well, incubated at 37 °C 2 In the hour, wash it again with PBS.
  • the DC cells on the 7th day of culture were collected, centrifuged, resuspended in 10% FBS RPMI 1640 medium, counted, formulated into 5 ⁇ 10 4 cells/ml, and added to the above anti-CD3 coated 96 by 90 ul/well. - In the orifice plate.
  • PBMC cells from different volunteers were taken, counted, and formulated into 5 ⁇ 10 5 cells/ml, and added to the 96-well plate coated with the above-mentioned anti-CD3 and plated with DC cells at 90 ul/well.
  • the sample to be tested was prepared in proportion to PBS, including a negative antibody, a control antibody, a PD-1 antibody (cloned according to the sequence disclosed by Opdivo, cloned according to Example 1 and purified by the expression), and added to the above 96-well plate at 20 ul/well.
  • the concentration of the antibody to be tested in the 200 ul system is the desired concentration gradient.
  • Control group 90 ul PBMC cells + 90 ul DC + 20 ul PBS.
  • the cell culture plate After incubating the cell culture plate at 37 ° C for 6 days in a 5% CO 2 incubator, the cell culture plate was taken out, centrifuged at 3000 rpm for 10 min, and 150 ⁇ l of the supernatant was taken out per well for detection of IFN- ⁇ .
  • the extracted cell culture supernatant was diluted 25 times (different dilutions of different experiments) and added to the microplate (100 ul/well); the standard was diluted with the common dilution of the sample to a different concentration gradient: 1000 pg/ml 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, 100 ⁇ l per well; blank wells plus standard dilutions.
  • the ab32 antibody concentration was increased to 5 ug/ml
  • the IFN- ⁇ secreted by T cells increased to 38.9% and 30.1%. That is, the antibody ab32 (5 ug/ml) and the PD-1 antibody (3 ug/ml) of the present invention can increase the secretion of IFN- ⁇ by T cells, and the increase rate is (30.1-22.8)/22.8, that is, 32%.
  • the binding activity of the antibody of the present invention and non-human Tim-3 was examined by the method of Example 3.
  • the non-human Tim-3 proteins used included macaque (Macaca) and marmoset Tim-3 proteins (see Table 1 for expression of each protein), and the results are shown in Figures 4a, 4b.
  • the data of the present invention shows that the antibody discovered by the invention has unique binding activity, fast binding and slow dissociation characteristics, humanized and fully humanized low immunogenicity, and activates the excellent activity of human blood cells to kill tumor cells.
  • the PD-1 antibody alone and in combination can enhance PD-1 activated T cell (MLR) functional activity, etc., making it suitable for human Tim-3 target antibody drug development, and as an excellent drug candidate, tumors can be treated alone or in combination.
  • MLR PD-1 activated T cell

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Abstract

L'invention concerne un anticorps TIM-3, un fragment de liaison à l'antigène de celui-ci et une utilisation médicale associée. Plus particulièrement, l'invention concerne un anticorps murin, un anticorps chimérique, un anticorps humanisé, un anticorps complètement humanisé, un anticorps à liaison rapide et à dissociation lente contenant des régions CDR de l'anticorps TIM-3, un anticorps TIM-3 qui est excellent en termes d'activation de l'activité fonctionnelle des cellules sanguines humaines, une composition pharmaceutique contenant l'anticorps TIM-3 humain et un fragment de liaison à l'antigène de celui-ci, ainsi que l'utilisation de celui-ci en tant que médicament anticancéreux. En particulier, l'invention concerne une composition pharmaceutique de l'anticorps TIM-3 en combinaison avec un anticorps PD-1, et son utilisation dans le traitement de tumeurs.
PCT/CN2019/078313 2018-03-15 2019-03-15 Molécule d'anticorps complètement humanisé contre tim-3, fragment de liaison à l'antigène et utilisation médicale associée WO2019174637A1 (fr)

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CN103079644A (zh) * 2010-06-11 2013-05-01 协和发酵麒麟株式会社 抗tim-3抗体
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CN103079644A (zh) * 2010-06-11 2013-05-01 协和发酵麒麟株式会社 抗tim-3抗体
CN102492038A (zh) * 2011-12-09 2012-06-13 中国人民解放军军事医学科学院基础医学研究所 抗人Tim-3的中和性单克隆抗体L3D及其用途
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CN107405397A (zh) * 2014-10-27 2017-11-28 新加坡科技研究局 抗tim‑3抗体
CN104592388A (zh) * 2015-03-02 2015-05-06 中国人民解放军总医院 一种抗人Tim-3的单克隆抗体的抗原结合部分
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WO2023217268A1 (fr) * 2022-05-13 2023-11-16 正大天晴药业集团南京顺欣制药有限公司 Combinaison médicamenteuse d'anticorps anti-tim-3 et d'anticorps anti-pd-1

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