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WO2022228183A1 - 抗siglec15抗体及其制备方法和用途 - Google Patents

抗siglec15抗体及其制备方法和用途 Download PDF

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WO2022228183A1
WO2022228183A1 PCT/CN2022/087405 CN2022087405W WO2022228183A1 WO 2022228183 A1 WO2022228183 A1 WO 2022228183A1 CN 2022087405 W CN2022087405 W CN 2022087405W WO 2022228183 A1 WO2022228183 A1 WO 2022228183A1
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seq
antibody
siglec15
amino acid
acid sequence
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PCT/CN2022/087405
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English (en)
French (fr)
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李朝辉
刘文慧
方杰
吴敏
刘雯珲
吕裕斌
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杭州邦顺制药有限公司
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Publication of WO2022228183A1 publication Critical patent/WO2022228183A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Definitions

  • the present invention relates to the field of biomedicine, in particular to an antibody against Sigelc15 or an antigen-binding fragment thereof.
  • Sialic acid-binding Ig-like lectins are members of a class of immunoglobulin superfamily, belonging to a class of type I transmembrane proteins, mainly including sialic acid-binding N-terminal IgV Domains, IgC2 variable domain domains, transmembrane domains, and cytoplasmic domains mediate cell-cell or pathogenic interactions by recognizing sialic acid-containing sugar chain structures.
  • the Siglec family is mainly divided into two categories.
  • One subtype mainly includes Siglecs with variable sequences related to CD33, such as Siglec7, Sglec8, Siglec9, etc., and the other is Siglecs with conserved sequences, including sialoadhesin, CD22, MAG and Siglec15; Recent studies have shown that members of the Siglec family play an important role in immune regulation in autoimmune diseases, inflammatory responses and tumorigenesis, thus becoming targets for drug therapy.
  • Siglec15 protein is an important immunosuppressive molecule.
  • Siglec15 inhibits the proliferation of immune T cells by binding to ligands on immune T cells.
  • Siglec15 antibody relieves immune T cells by blocking the binding of Sigelc15 to its ligands. Inhibition, which can serve as a potential target for tumor immune normalization strategies (Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy, Jun Wang, et al., nature medicine 4(25):656-666(2019) ).
  • Siglec15 mRNA is rarely expressed in most normal human tissues and various immune cell subsets, but TCGA data show that Siglec15 mRNA has a broad expression profile in human tumors, including colon, endometrioid, and thyroid cancers , bladder cancer, kidney cancer, lung cancer and liver cancer, as well as tumor-infiltrating macrophages can also detect the expression of Siglec15.
  • Siglec15 has broad application prospects in the clinic, so it is urgent to develop Siglec15 antibody to meet the therapeutic needs of patients in this field.
  • Chinese patent CN110035769A discloses an antibody or an antigen-binding fragment thereof that specifically binds to Siglec15.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, comprising:
  • a heavy chain variable region comprises at least one of the following HCDRs:
  • HCDR1 its amino acid sequence is as shown in SEQ ID NO:1, or comprises the aminoacid sequence shown in SEQ ID NO:1;
  • HCDR2 its amino acid sequence is as shown in SEQ ID NO:2 or SEQ ID NO:13, or comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:13;
  • HCDR3 whose amino acid sequence is shown in SEQ ID NO:3, or comprises the amino acid sequence shown in SEQ ID NO:3;
  • a light chain variable region comprises at least one of the following LCDRs:
  • LCDR1 whose amino acid sequence is shown in SEQ ID NO:4 or SEQ ID NO:14, or comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:14;
  • LCDR2 its amino acid sequence is as shown in SEQ ID NO:5, or comprises the amino acid sequence shown in SEQ ID NO:5;
  • LCDR3 whose amino acid sequence is shown in SEQ ID NO:6, or comprises the amino acid sequence shown in SEQ ID NO:6.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO: 1.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises HCDR2 whose amino acid sequence is shown in SEQ ID NO: 2.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises HCDR2 whose amino acid sequence is shown in SEQ ID NO: 13.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises HCDR3 whose amino acid sequence is shown in SEQ ID NO:3.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody light chain variable region comprises LCDR1 whose amino acid sequence is shown in SEQ ID NO:4.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody light chain variable region comprises LCDR1 whose amino acid sequence is shown in SEQ ID NO: 14.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody light chain variable region comprises LCDR2 whose amino acid sequence is shown in SEQ ID NO:5.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody light chain variable region comprises LCDR3 whose amino acid sequence is shown in SEQ ID NO:6.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, respectively and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:3.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 13, respectively and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:3.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody light chain variable region comprises amino acid sequences such as SEQ ID NO:4, SEQ ID NO:5, respectively and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:6.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody light chain variable region comprises amino acid sequences such as SEQ ID NO: 14, SEQ ID NO: 5, respectively and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:6.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, respectively and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3; and wherein said antibody light chain variable region comprises amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively of LCDR1, LCDR2 and LCDR3.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, respectively and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3; and wherein said antibody light chain variable region comprises amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 6, respectively of LCDR1, LCDR2 and LCDR3.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 13, respectively and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3; and wherein said antibody light chain variable region comprises amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively of LCDR1, LCDR2 and LCDR3.
  • the present invention provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 13, respectively and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3; and wherein said antibody light chain variable region comprises amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO: 6, respectively of LCDR1, LCDR2 and LCDR3.
  • the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 7, or the same as SEQ ID NO: 7 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and/or the light chain variable region amino acid sequence is shown in SEQ ID NO: 8 , or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:8.
  • the antibody is a murine antibody or a fragment thereof.
  • the murine antibody or its fragment heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 2 and SEQ ID NO: 3; and the antibody light chain variable region described therein comprises amino acid sequences such as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID, respectively LCDR1, LCDR2 and LCDR3 shown in NO:6.
  • the heavy chain variable region thereof further comprises the heavy chain FR region of murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • the murine antibody or fragment thereof provided according to the present invention further comprises a heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • variable region of the antibody light chain further comprises the light chain FR region of a murine ⁇ , ⁇ chain or a variant thereof.
  • the murine antibody or fragment thereof provided according to the present invention further comprises a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof.
  • the antibody is a chimeric antibody or a fragment thereof.
  • the anti-Siglec15 chimeric antibody or fragment thereof provided according to the present invention further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof, preferably a human ⁇ light chain constant Area.
  • the anti-Siglec15 chimeric antibody or fragment thereof provided according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human source IgG1 or IgG4 heavy chain constant regions.
  • the heavy chain amino acid sequence of the antibody is as shown in SEQ ID NO: 9, or has at least SEQ ID NO: 9 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;
  • the light chain amino acid sequence of the antibody is as shown in SEQ ID NO:10, or with SEQ ID NO:10 have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the antibody is a humanized antibody or a fragment thereof.
  • the heavy chain variable region thereof further comprises the heavy chain FR of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof regions, preferably comprising human IgG1 or IgG4 heavy chain FR regions.
  • the heavy chain variable region sequence is selected from any of the sequences shown in SEQ ID NO: 15, 17, 19 or 22 One, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 15, 17, 19 or 22.
  • the anti-Siglec15 humanized antibody or fragment thereof provided according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human Source IgG1 or IgG4 heavy chain constant region, more preferably, the amino acid sequence of the human IgG1 heavy chain constant region is shown in SEQ ID NO: 23, or the amino acid sequence of the human IgG4 heavy chain constant region is shown in SEQ ID NO: 25 shown.
  • its light chain variable region further comprises the light chain FR region of human ⁇ , ⁇ chain or its variant, preferably Human kappa light chain FR region.
  • its light chain variable region sequence is selected from the sequence shown in SEQ ID NO: 16, 18, 20 or 21. Any one, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 16, 18, 20 or 21.
  • the anti-Siglec15 humanized antibody or fragment thereof provided according to the present invention further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof, preferably a human ⁇ light chain
  • a constant region more preferably a human kappa light chain constant region, is shown in SEQ ID NO:24.
  • its heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or the same as SEQ ID NO: 15 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity
  • its light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or Has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 16.
  • its heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, or the same as SEQ ID NO: 17 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
  • its light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, or Has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18.
  • its heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19, or the same as SEQ ID NO: 19 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity
  • its light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20, or Has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:20.
  • its heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, or the same as SEQ ID NO: 17 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
  • its light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20, or Has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:20.
  • its heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, or the same as SEQ ID NO: 17 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
  • its light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21, or Has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:21.
  • its heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, or the same as SEQ ID NO: 22 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
  • its light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20, or Has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:20.
  • the light chain variant thereof preferably has 0-10 amino acid changes in the light chain variable region.
  • the heavy chain variant thereof preferably has 0-10 amino acid changes in the variable region of the heavy chain.
  • the antigen-binding fragment is selected from Fab, Fv, scFv or F(ab')2.
  • the present invention further provides a kind of biological material, and this biological material can be:
  • the host cell is preferably a human embryonic kidney 293 cell or a Chinese hamster ovary cell.
  • the present invention further provides a method for producing an anti-Siglec15 antibody or an antigen-binding fragment thereof, comprising the steps of: culturing the host cell as described above; further, separating the antibody from the obtained culture; and purifying the antibody .
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-Siglec15 antibody or antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable excipient, diluent or carrier.
  • the present invention further provides a detection or diagnostic kit, which contains the anti-Siglec15 antibody or antigen-binding fragment thereof according to the present invention, for the detection, diagnosis, and prognosis of Siglec15 or Siglec15-mediated diseases or disorders.
  • the present invention further provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, biological materials (such as DNA molecules, expression vectors, host cells and cultures thereof) according to the present invention prepared for the treatment or prevention of Siglec15-mediated diseases or use in medicines for disorders.
  • biological materials such as DNA molecules, expression vectors, host cells and cultures thereof
  • the present invention further provides an anti-Siglec15 antibody or an antigen-binding fragment thereof, biological materials (such as DNA molecules, expression vectors, host cells and cultures thereof), compositions and kits according to the present invention in detection, diagnosis, and prognosis Use of Siglec15 or a disease or disorder mediated by Siglec15.
  • biological materials such as DNA molecules, expression vectors, host cells and cultures thereof
  • compositions and kits according to the present invention in detection, diagnosis, and prognosis Use of Siglec15 or a disease or disorder mediated by Siglec15.
  • the present invention further provides a method for the treatment and prevention of Siglec15-mediated diseases or disorders, the method comprising administering to a patient in need thereof a therapeutically effective amount of an anti-Siglec15 antibody or antigen-binding fragment thereof, biological material (such as DNA) according to the present invention molecules, expression vectors, host cells and cultures thereof) or pharmaceutical compositions comprising the anti-Siglec15 antibody or antigen-binding fragment thereof.
  • biological material such as DNA
  • the disease or condition mediated by Siglec15 according to the present invention is a cancer expressing Siglec15, such as colorectal cancer, endometrioid cancer, thyroid cancer, bladder cancer, kidney cancer, lung cancer, head and neck cancer, breast cancer, ovarian cancer or liver cancer.
  • a cancer expressing Siglec15 such as colorectal cancer, endometrioid cancer, thyroid cancer, bladder cancer, kidney cancer, lung cancer, head and neck cancer, breast cancer, ovarian cancer or liver cancer.
  • the anti-Siglec15 antibody and the antigen-binding fragment thereof of the present invention have higher binding activity to the Siglec15 protein, have a better function of reversing the inhibition of immune cell proliferation in vitro, and exhibit a better effect of inhibiting tumor growth.
  • Figure 1 Results of the reversal of hS15-Fc-mediated suppression of human T cells by the murine antibodies of the present invention.
  • Figure 2 Results of reversal of hS15-Fc mediated suppression of human T cells by chimeric antibodies of the present invention.
  • Figure 3 Antitumor activity of the chimeric antibodies of the present invention on the MC38-human Siglec15 colorectal cancer model.
  • Figure 4 Binding ability of the humanized antibody of the present invention to CHO-human Siglec15 cells.
  • Figure 5 Binding ability of the humanized antibody of the present invention to MC38-mouseSiglec15 cells.
  • Figure 6 The results of reversing hS15-Fc-mediated inhibition of human T cells by the IgG4-type humanized antibody of the present invention, the concentration indicated in the figure represents the antibody concentration.
  • Fig. 7 The result of reversing hS15-Fc-mediated inhibition of human T cells by the IgG1-type humanized antibody of the present invention, the concentration indicated in the figure represents the antibody concentration.
  • the antibody in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain, ⁇ chain.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain of the present invention may further comprise a light chain constant region comprising the light chain constant region of a human or murine ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises the heavy chain constant of human or murine IgG1, 2, 3, 4 or a variant thereof Area.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions. The order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.
  • the number and position of CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3), or the numbering rules of Kabat and Chothia.
  • murine antibody in the present invention is a monoclonal antibody against human Siglec15 prepared according to the knowledge and skills in the art. In preparation, test subjects (mice) are injected with Siglec15 antigen, and then antibodies with desired sequences or functional properties are isolated and expressed.
  • the murine Siglec15 antibody or its antigen-binding fragment may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a murine IgG1, IgG2 , the heavy chain constant region of IgG3 or IgG4 or a variant thereof.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To build a chimeric antibody select a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into the vector, and finally the chimeric antibody molecule is expressed in the eukaryotic industrial system or the prokaryotic industrial system.
  • the antibody light chain variable region of the anti-Siglec15 chimeric antibody further comprises a light chain FR region of a murine ⁇ , ⁇ chain or a variant thereof, and the antibody light chain variable region sequence As shown in SEQ ID NO:8.
  • the antibody heavy chain variable region of the described anti-Siglec15 chimeric antibody further comprises the heavy chain FR region of murine IgG1, IgG2, IgG3, IgG4 or its variant, and the antibody heavy chain variable region sequence is as shown in SEQ ID NO: 7. Show.
  • the constant region of the human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising the heavy chain constant regions of human IgG1 or IgG4.
  • the light chain constant region of a human antibody may be selected from the light chain constant regions of human kappa, lambda chains or variants thereof, preferably comprising a human kappa light chain constant region.
  • humanized antibody also known as CDR-grafted antibody, refers to the addition of CDR sequences of non-human origin (eg, mice) without significantly affecting the antigen-binding properties.
  • Antibody variable region frameworks grafted into humans. Humanized antibodies can overcome the disadvantage of strong immune responses induced by chimeric antibodies because they carry a large number of mouse protein components.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al. People, 1991 Sequences of Proteins of Immunological Interest, 5th ed.
  • the variable regions of the human antibody may be subjected to minimal reverse mutations to maintain activity.
  • the "antigen-binding fragment” described in the present invention refers to Fab fragments, Fab' fragments, F(ab') 2 fragments with antigen-binding activity, and Fv fragments and scFv fragments that bind to human Siglec15, including those described in the present invention.
  • Fv fragments contain antibody heavy and light chain variable regions, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Different linkers can also be used to link the two antibody variable regions into a single polypeptide chain, called a single chain antibody or single chain Fv (scFv).
  • Fab fragments are monovalent fragments composed of VL, VH, CL, and CH1 domains.
  • F(ab')2 is a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region.
  • mice can be immunized with human Siglec15 or a fragment thereof, and the resulting antibody can be renatured, purified, and amino acid sequenced by conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibody or mAb in the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • Antibodies or antigen-binding fragments can be obtained recombinantly using, eg, hybridoma technology, recombinant technology, phage display technology, synthetic technology (eg, CDR-grafting), or other existing techniques.
  • administering and “treatment” when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to the interaction of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with the animal, human, subject, or biological fluid. contact with subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administeristering” and “treating” also mean in vitro and ex vivo treatment of eg cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the anti-Siglec15 antibodies or antigen-binding fragments thereof of the invention, to a patient with one or more disease symptoms for which the Therapeutic agents have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a patient or population being treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinical extent.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by physicians or other health care professionals to assess the severity or progression of the symptoms, such as according to statistical tests known in the art such as the Student's t-test , Chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test to determine.
  • statistical tests known in the art such as the Student's t-test , Chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test to determine.
  • Constant modification refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) without changing biological activity of the protein.
  • Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • the variant of the antibody light chain or heavy chain is the "conservative modification” or “conservative substitution or substitution” of 0-10 amino acids in the light chain or heavy chain, which can be expected by those skilled in the art
  • the variant has substantially the same activity as before the modification or substitution.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a medical condition or symptom.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is to be produced outside the organism, cell, or human body depending on the context.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the context.
  • Sequence identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine in two DNA molecules, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, when sequences are optimally aligned, two sequences are 60% identical if 6 of 10 positions in the sequences are matched or homologous. In general, comparisons are made when two sequences are aligned for the greatest percent identity.
  • One skilled in the art can determine the number of bases or amino acids that change as represented by the percent sequence identity.
  • progeny As used herein, the expressions "cell”, “cell line” and “cell strain” are used interchangeably and all such designations include progeny. Thus, “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutation, all progeny may not be exactly the same in DNA content, including mutant progeny that have the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clear from the context.
  • “Pharmaceutical composition” means a mixture comprising one or more of the anti-Siglec15 antibodies or antigen-binding fragments thereof described herein, together with other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • the GenBank accession number of Siglec15 protein or S15 protein or Siglec15 antigen is NM_213602.
  • the present invention uses the extracellular region (20-263) of human Siglec15 as an antigen to immunize mice, so that the mice can generate an immune response to produce a murine antibody against Siglec15.
  • hS15-his refers to a fusion protein in which 6 histidines are linked to the C-terminus of the extracellular domain of human Siglec15 protein.
  • hS15-Fc refers to a fusion protein in which the Fc fragment of the human IgG1 constant region is linked to the C-terminus of the extracellular region of the human Siglec15 protein.
  • hS15-Fc-mediated inhibition of human T cells refers to the phenomenon that human Siglec15 protein inhibits the proliferation of immune T cells by binding to ligands on immune T cells.
  • anti-human CD3 antibody (clone OKT3, biolegend) refers to an antibody against the CD3 antigen on immune T cells, which is used to activate the proliferation of T cells.
  • the MC38-humanSiglec15 in vivo tumor model is a mouse colorectal cancer model established on C57BL/6N mice.
  • the cDNA sequence encoding human Siglec15 (GenBank accession number NM_213602) was transfected into MC38 blank cells to obtain a stable cell line MC38-humanSiglec15 (MC38-hS15) overexpressing human Siglec15.
  • DNA sequences encoding the CDRs, variable regions or light and heavy chains of the anti-Siglec15 antibody involved in the present invention can be designed according to the corresponding amino acid sequences, which is a routine technique in the art.
  • the present invention is further described below in conjunction with the embodiments, but these embodiments do not limit the scope of the present invention, and those skilled in the art can understand other advantages and effects of the present invention from the contents disclosed in this specification.
  • the experimental method that does not indicate specific conditions in the embodiment of the present invention, usually according to conventional conditions, such as the antibody technology experimental manual of Cold Spring Harbor, molecular cloning manual; Or according to the conditions suggested by raw material or commodity manufacturers. Reagents with no specific source indicated are conventional reagents purchased in the market.
  • the hybridoma antibody against human Siglec15 was prepared by conventional animal immunization and fusion methods: the DNA encoding human Siglec15 was inserted into the expression vector, and Expi293F cells were transfected by transient transfection (Lifetechnologies, 12338-018), After further purification, the immunization antigen human Siglec15 was obtained. 500 ⁇ g of human Siglec15 antigen and an equal volume of Freund's complete adjuvant (Sigma-Aldrich) were mixed and emulsified at a ratio of 1:1, and the emulsion was subcutaneously immunized with female C57BL/6N mice (Beijing Weitonglihua).
  • mice were then boosted on days 14 and 28 by subcutaneous injection of a 1:1 mixture of 250 ⁇ g of an emulsion of human Siglec15 protein and incomplete Freund's adjuvant (Sigma-Aldrich).
  • the antibody titer of mouse serum was determined by enzyme-linked immunosorbent assay (Elisa): hS15-his at a concentration of 0.5 ⁇ g/mL was added to a 96-well plate at a volume of 100 ⁇ L/well 4 °C coated overnight.
  • PBST phosphate buffered saline containing 0.05% Tween 20
  • BSA bovine serum albumin
  • Boosted mice were sacrificed, spleens were extracted and homogenized to generate single cell suspensions, and myeloma cell (SP2/0) (SGST) single cell suspensions were prepared.
  • Splenocytes were fused with SP2/0 mouse myeloma cells using electrofusion.
  • the fused cells were resuspended in complete medium containing hybridoma selection agent DMEM+20% FBS+HAT (Gibco) and seeded into 96-well plates at 200 ⁇ L/well.
  • the 96-well plate was cultured in an incubator at 37° C. and 5% CO 2 for 6-7 days, and the cell supernatant was taken, and the binding of mouse-derived antibody to Siglec15 protein was detected by ELISA.
  • Selected preferred hybridoma clones were subcloned using limiting dilution. After culturing the hybridoma cells in 37°C 5% CO 2 for 1 week, the supernatant was subjected to ELISA binding detection, and the murine antibody E05 against Siglec15 was screened according to the binding and function of the murine antibody to Siglec15 protein.
  • Sequencing the variable region of mouse antibody E05 extract the mRNA of hybridoma cells, reverse transcribed into cDNA, carry out PCR with universal primers, sequence and analyze the DNA products obtained by PCR, and then translate them into amino acid sequences, and analyze the variable regions.
  • the CDR region was analyzed using the Kabat rule, wherein the heavy chain CDR1 was increased by 5 amino acids according to the IMGT principle, and the results are shown in Table 1.
  • PBMC peripheral blood lymphocytes
  • the unbound OKT3 supernatant was aspirated and washed three times with PBS; the fusion protein of soluble human Siglec15 and human Fc (hS15-Fc) 50 ⁇ L/well (final concentration 1.5 ⁇ g/ml) and E05 antibody 50 ⁇ l/well ( 2 ⁇ g/ml) were mixed and added to 96-well plates, and control wells without E05 (OKT3+hS15-Fc 1.5 ⁇ g/ml) and without hS15-Fc and E05 antibodies (OKT3) were set; PBMCs were collected and washed with PBS After one time, resuspend in PBS, add 5 ⁇ M fluorescent dye CFSE (hydroxyfluorescein diacetate succinimidyl ester) and incubate at 37°C for 15 minutes.
  • CFSE hydroxyfluorescein diacetate succinimidyl ester
  • Complete medium was resuspended to a cell density of 3E06, 50 ⁇ L/well was added to a 96-well plate, and the 96-well plate was incubated in a 37°C, 5% CO 2 incubator for 72 hours. The cells were transferred to a round-bottomed 96-well plate, washed twice with PBS buffer, and analyzed for the proliferation effect of PBMC by flow cytometry.
  • mouse-derived antibody 5G12 (mIgG1) was prepared with reference to the method described in Chinese patent CN110035769A, which was used as a positive control group to conduct a control experiment.
  • the mouse-derived antibody E05 can significantly block the proliferation inhibition effect of hS15-Fc on T cells, and the blocking effect is better than that of the positive control antibody 5G12 (in Figure 1, "without OKT3" is the negative control, i.e. only PBMC cells).
  • the heavy and light chain variable regions of murine antibody E05 were directionally inserted into the expression vector pcDNA3.4 (GenScript) containing the signal peptide and human IgG1 constant region, and the signal peptide and human light chain kappa constant region, respectively.
  • Antibody name heavy chain light chain ChE05 SEQ ID NO: 9 SEQ ID NO: 10
  • a humanized 5G12 antibody (h5G12) was prepared as a positive control group for a control test, wherein CN110035769A only disclosed the variable region of h5G12, and the inventors of the present application compared it with human.
  • the full-length sequence of the humanized 5G12 antibody was prepared by combining the IgG1 constant regions.
  • the Siglec15 chimeric antibody ChE05 can better block the proliferation inhibitory effect of hS15-Fc on T cells, and its blocking effect is better than that of the positive control antibody h5G12.
  • Example 5 The effect of Siglec15 chimeric antibody in inhibiting tumor growth in colorectal cancer model in vivo
  • the in vivo immunomodulatory activity of the antibodies of the present invention was measured using the MC38-humanSiglec15 in vivo tumor model.
  • the cDNA sequence encoding human Siglec15 was transfected into MC38 blank cells to obtain a stable cell line MC38-humanSiglec15 (MC38-hS15) overexpressing human Siglec15.
  • Each mouse was injected with 10 mg/kg of antibody, twice a week, for a total of 5 times; 3 times a week
  • the body weight and tumor size of the mice were measured, and the tumor volume was calculated according to (length ⁇ width ⁇ 2)*0.5, and the average tumor volume of each group of mice was taken as the tumor growth curve graph.
  • Example 4 Using the h5G12 antibody in Example 4 as a positive control group, a control experiment was carried out.
  • the Siglec15 chimeric antibody ChE05 inhibited the growth of MC38-hS15 tumors significantly better than the control group h5G12.
  • Humanization is carried out on the basis of obtaining the variable region of the light chain and the variable region of the heavy chain of the murine antibody obtained in the above example.
  • the 6 CDRs of the heavy chain and light chain of the mouse antibody were grafted onto a human template with high similarity to the mouse FR region.
  • the human template was obtained from the PDB database through BLAST and had a high similarity to the mouse antibody sequence.
  • the germline sequence of wherein the light chain variable region template is human germline light chain IGKV4-1*01, and the heavy chain variable region template is human germline heavy chain IGHV3-7*01.
  • the CDR-grafted antibody was subjected to homology modeling to predict the key amino acids in the murine anti-FR region that may determine the structure of the antibody.
  • post-translational modification (PTM) analysis of the grafted sequence showed that the variable region of the antibody light chain CDR1 and The CDR2 of the variable region of the heavy chain contains an isomerization modification site.
  • the method of back mutation is used to back-mutate the amino acids of individual human templates in the FR region back to mouse amino acids. and screening to finally obtain humanized antibodies.
  • the amino acid sequence of the back-mutated heavy chain CDR2 is SEQ ID NO: 13
  • the amino acid sequence of the back-mutated light chain CDR1 is: SEQ ID NO: 14.
  • sequence the obtained humanized antibody synthesize cDNA according to the amino acid sequence of the light chain variable region and heavy chain variable region of the humanized antibody, and insert the signal peptide and human IgG1 or IgG4 (S228P) heavy chain constant region,
  • the expression plasmid of the full-length antibody was obtained from the pcDNA3.4 expression vector (GenScript) containing the signal peptide and the constant region of human light chain kappa.
  • the heavy chain and light chain expression plasmids were co-transfected into Expi293F cells, and the supernatant was harvested after culturing for 6-7 days for Protein A purification to obtain the humanized antibody of the present invention.
  • Humanized antibody affinity determination was performed using Biacore T200 Biomacromolecule Interaction Instrument (GE Healthcare). Conjugate anti-human IgG-Fc antibody on the chip, use anti-human IgG antibody to capture Siglec15 humanized antibody, use antigen Siglec15-his as mobile phase, use 6 concentration gradients (20, 10, 5, 2.5, 1.25, 0.625 , 0.3125 nM), followed by a 20 sec association step and a 360 sec dissociation step. Regeneration was performed using 10 mM glycine-hydrochloric acid buffer for 60 s; data were processed using Biacore T200 data analysis software 3.1. The test results are shown in Table 4.
  • Example 8 FACS determination of the binding of Siglec15 humanized antibody to CHO-hS15 and MC38-mS15 cells
  • the cDNA sequences encoding human Siglec15 and mouse Siglec15 were transfected into CHO and MC38 blank cells, respectively, to obtain stable cell lines CHO-HumanSiglec15 (CHO-hS15) and overexpressing human Siglec15 and mouse Siglec15.
  • CHO-hS15 CHO-hS15
  • MC38-mouseSiglec15 MC38-mS15
  • CHO-hS15 and MC38-mS15 were resuspended in PBS to a cell density of 4E06.
  • Cells were added to a 96-well plate at 50 ⁇ L/well, and 50 ⁇ L of Siglec15 humanized antibody was added at final concentrations ranging from 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 ⁇ g/ml. Cells were incubated at 4°C for 30 minutes and then washed twice with PBS buffer. Cells were resuspended with 100 ⁇ L of goat anti-human IgG-FITC secondary antibody (Jackson Immunoresearch, 109-545-003) 1:200 dilution, incubated at 4°C for 30 min and washed twice with PBS buffer, and analyzed by flow cytometry , the results are shown in Figures 4 and 5.
  • PBMC peripheral blood lymphocytes
  • IgG1 and IgG4 50 ⁇ l/well (20, 6.67, 2.22, 0.74, 0.24 ⁇ g/ml) were mixed and added to the 96-well plate, and the S15 humanized antibody (OKT3+hS15-Fc 1.5 ⁇ g/ml) and Control wells without hS15-Fc and S15 humanized antibody (OKT3); PBMCs were collected, washed once with PBS, resuspended in PBS, added 5 ⁇ M CFSE and incubated at 37°C for 15 minutes, and washed twice with PBS after incubation , resuspend to the cell density of 3E06 with complete medium of 1640+10% FBS, add 50 ⁇ L/well to 96-well plate, and incubate 96-well plate in 37°C CO 2 incubator for 72 hours. The cells were transferred to a 96-well round bottom plate, and after washing twice with PBS buffer, the proliferation effect of PBMC was analyzed by flow cytometry.
  • the Siglec15 humanized antibody of the present invention can better block the proliferation inhibitory effect of hS15-Fc on T cells, and the blocking activity is better than that of the positive control h5G12 antibody ( Figure 6 and Figure 7 ).
  • the "without OKT3" group is a negative control; in Figure 6, "ChE05-4, h5G12-4" is obtained by replacing the heavy chain constant region IgG1 of ChE05 and h5G12 with IgG4).
  • Example 10 The effect of Siglec15 humanized antibody on tumor growth inhibition in vivo
  • Example 5 the MC38-humanSiglec15 in vivo tumor model was used to measure the in vivo immunomodulatory activity of the Siglec15 humanized antibody of the present invention.
  • the effect of tumor inhibition is shown in Table 5.

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Abstract

一种抗Siglec15抗体、其抗原结合片段及其医药用途，包含所述抗体CDR区的嵌合抗体、人源化抗体、包含抗Siglec15抗体及其抗原结合片段的药物组合物，以及所述抗体在制备用于治疗疾病或病症的药物中的用途。

Description

抗Siglec15抗体及其制备方法和用途 技术领域
本发明涉及生物医药领域，具体的涉及一种针对Sigelc15的抗体或其抗原结合片段。
背景技术
唾液酸结合性免疫球蛋白样凝集素(sialic acid-binding Ig-like lectins，Siglecs)是一类免疫球蛋白超家族成员，属于一类I型跨膜蛋白，主要包括唾液酸结合性N端IgV结构域、IgC2可变区结构域、跨膜区和胞质区，可通过识别含有唾液酸的糖链结构，介导细胞与细胞或病原体间的相互作用。Siglec家族主要分为两类，一类亚型主要包括CD33相关的序列可变的Siglecs，如Siglec7、Sglec8、Siglec9等，另一类是序列保守的Siglecs，包括唾液酸黏附素、CD22、MAG和Siglec15；近年来的研究表明Siglec家族成员在自身免疫病、炎症反应以及肿瘤的发生中发挥重要的免疫调控作用，从而成为药物治疗的靶标。
最近的研究发现Siglec15蛋白是一个重要的免疫抑制分子，Siglec15通过与免疫T细胞上的配体结合从而抑制免疫T细胞的增殖，Siglec15抗体通过阻断Sigelc15与其配体的结合从而解除免疫T细胞的抑制，其可作为肿瘤免疫正常化策略的潜在靶点(Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy，Jun wang,et al.,nature medicine 4(25):656-666(2019))。Siglec15 mRNA在大多数正常人体组织和各种免疫细胞亚群中均罕见不表达，但是TCGA数据显示Siglec15 mRNA在人类的肿瘤上有广泛的表达谱，包括结肠癌、子宫内膜样癌、甲状腺癌、膀胱癌、肾癌、肺癌和肝癌等，以及肿瘤浸润的巨噬细胞上也能检测到Siglec15的表达。Siglec15在临床中有着广泛的应用前景，因此亟待开发Siglec15抗体以满足本领域病人的治疗需求。
中国专利CN110035769A公开了一种特异性结合Siglec15的抗体或其抗原结合片段。
目前，针对Siglec15的抗体及其效果有待进一步提高和完善。
发明内容
为了解决现有技术中存在的问题，本发明提供了一种抗Siglec15抗体或其抗原结合片段。
本发明提供一种抗Siglec15抗体或其抗原结合片段，其包含：
重链可变区，所述的重链可变区包含至少1个如下HCDR：
HCDR1，其氨基酸序列如SEQ ID NO:1所示，或包含SEQ ID NO:1所示氨基酸序列；
HCDR2，其氨基酸序列如SEQ ID NO:2或SEQ ID NO:13所示，或包含SEQ ID NO:2或SEQ ID NO:13所示氨基酸序列；
HCDR3，其氨基酸序列如SEQ ID NO:3所示，或包含SEQ ID NO:3所示氨基酸序列；和
轻链可变区，所述的轻链可变区包含至少1个如下LCDR：
LCDR1，其氨基酸序列如SEQ ID NO:4或SEQ ID NO:14所示，或包含SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列；
LCDR2，其氨基酸序列如SEQ ID NO:5所示，或包含SEQ ID NO:5所示氨基酸序列；
LCDR3，其氨基酸序列如SEQ ID NO:6所示，或包含SEQ ID NO:6所示氨基酸序列。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列如SEQ ID NO:1所示的HCDR1。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列如SEQ ID NO:2所示的HCDR2。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列如SEQ ID NO:13所示的HCDR2。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列如SEQ ID NO:3所示的HCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体轻链可变区包含氨基酸序列如SEQ ID NO:4所示的LCDR1。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体轻链可变区包含氨基酸序列如SEQ ID NO:14所示的LCDR1。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体轻链可变区包含氨基酸序列如SEQ ID NO:5所示的LCDR2。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体轻链可变区包含氨基酸序列如SEQ ID NO:6所示的LCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:13和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3 所示的HCDR1、HCDR2和HCDR3；且其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:13和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，本发明提供一种抗Siglec15抗体或其抗原结合片段，其中所述的抗体重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:13和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:14、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其抗原结合片段，其中所述的重链可变区氨基酸序列如SEQ ID NO:7所示，或与SEQ ID NO:7具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，和/或所述的轻链可变区氨基酸序列如SEQ ID NO:8所示，或与SEQ ID NO:8具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其抗原结合片段，其中所述的抗体为鼠源抗体或其片段。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其抗原结合片段，其中所述的鼠源抗体或其片段重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且其中所述的抗体轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在本发明一个优选的实施方案中，根据本发明提供的鼠源抗体或其片段，其重链可变区进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区。
在本发明一个优选的实施方案中，根据本发明提供的鼠源抗体或其片段，其进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
在本发明一个优选的实施方案中，根据本发明提供的鼠源抗体或其片段，其抗体轻链可变区进一步包含鼠源κ、λ链或其变体的轻链FR区。
在本发明一个优选的实施方案中，根据本发明提供的鼠源抗体或其片段，其进一步包含鼠源κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其抗原结合片段，其中所述的抗体为嵌合抗体或其片段。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15嵌合抗体或其片段，其进一步包含人源κ、λ链或其变体的轻链恒定区，优选人源κ轻链恒定区。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15嵌合抗体或其片段，其进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15嵌合抗体或其片段，所述抗体的重链氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；所述抗体的轻链氨基酸序列如SEQ ID NO:10所示，或与SEQ ID NO:10具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其抗原结合片段，其中所述的抗体为人源化抗体或其片段。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区，优选包含人源IgG1或IgG4重链FR区。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，重链可变区序列选自如SEQ ID NO:15、17、19或22所示序列中的任意一种，或与SEQ ID NO:15、17、19或22具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区，更优选所述的人源IgG1重链恒定区的氨基酸序列如SEQ ID NO：23所示，或所述的人源IgG4重链恒定区的氨基酸序列如SEQ ID NO：25所示。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其轻链可变区进一步包含人源κ、λ链或其变体的轻链FR区，优选人源κ轻链FR区。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其轻链可变区序列选自如SEQ ID NO:16、18、20或21所示序列中的任意一种，或与SEQ ID NO:16、18、20或21具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其进一步包含人源κ、λ链或其变体的轻链恒定区，优选人源κ轻链恒定区，更优选人源κ轻链恒定区的氨基酸序列如SEQ ID NO：24所示。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区包含如SEQ ID NO:15所示的氨基酸序列，或与SEQ ID NO:15具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区包含如SEQ ID NO:16所示的氨基酸序列，或与SEQ ID NO:16具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区包含如SEQ ID NO:17所示的氨基酸序列，或与SEQ ID NO:17具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区包含如SEQ ID NO:18所示的氨基酸序列，或与SEQ ID NO:18具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区包含如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区包含如SEQ ID NO:20所示的氨基酸序列，或与SEQ ID NO:20具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区包含如SEQ ID NO:17所示的氨基酸序列，或与SEQ ID NO:17具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区包含如SEQ ID NO:20所示的氨基酸序列，或与SEQ ID NO:20具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区包含如SEQ ID NO:17所示的氨基酸序列，或与SEQ ID NO:17具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区包含如SEQ ID NO:21所示的氨基酸序列，或与SEQ ID NO:21具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15人源化抗体或其片段，其重链可变区包含如SEQ ID NO:22所示的氨基酸序列，或与SEQ ID NO:22具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区包含如SEQ ID NO:20所示的氨基酸序列，或与SEQ ID NO:20具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
本领域技术人员根据上述的抗体重轻链可变区和恒定区的氨基酸序列，可以容易地知道上述抗体重轻链的全序列，并以此得知抗体序列全信息。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其片段，其轻链变体优选在轻链可变区有0-10个氨基酸变化。
在本发明一个优选的实施方案中，根据本发明提供的抗Siglec15抗体或其片段，其重链变体优选在重链可变区有0-10个氨基酸变化。
在本发明一个优选的实施方案中，根据本发明提供的Siglec15抗体或其抗原结合片段，其中所述的抗原结合片段选自Fab、Fv、scFv或F(ab’)2。
本发明进一步提供一种生物材料，该生物材料可以是：
(1)编码如上所述的抗Siglec15抗体或其抗原结合片段的DNA分子；所述DNA分子可以分别编码抗体的重链和轻链部分，本领域技术人员根据抗体或其抗原结合片段的氨基酸序列，可以推知DNA序列，并为其设置合适的表达元件，使DNA分子能够表达本发明的抗体或其抗原结合片段；
(2)含有如上所述的DNA分子的表达载体；
(3)含有如上所述的DNA分子或表达载体的宿主细胞，或上述宿主细胞经培养后获得的培养液、菌悬液等培养物。
在本发明一个优选的实施方案中，根据本发明提供的宿主细胞，其中所述的宿主细胞优选为人胚肾293细胞或中国仓鼠卵巢细胞。
本发明进一步提供一种生产抗Siglec15抗体或其抗原结合片段的方法，包括步骤：培养如上所述的宿主细胞；进一步地，还包括从获得的培养物中分离抗体；以及对所述抗体进行纯化。
本发明进一步提供一种药物组合物，其含有根据本发明所述的抗Siglec15抗体或其抗原结合片段和可药用的赋形剂、稀释剂或载体。
本发明进一步提供一种检测或诊断试剂盒，其含有根据本发明所述的抗Siglec15抗体或其抗原结合片段，用于检测、诊断、预后Siglec15或Siglec15介导的疾病或病症。
本发明进一步提供一种根据本发明所述的抗Siglec15抗体或其抗原结合片段、生物材料(如DNA分子、表达载体、宿主细胞及其培养物)在制备用于治疗或预防Siglec15介导的疾病或病症的药物中的用途。
本发明进一步提供一种根据本发明所述的抗Siglec15抗体或其抗原结合片段、生物材料(如DNA分子、表达载体、宿主细胞及其培养物)、组合物以及试剂盒在检测、诊断、预后Siglec15或Siglec15介导的疾病或病症中的用途。
本发明进一步提供一种治疗和预防Siglec15介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的根据本发明所述的抗Siglec15抗体或其抗原结合片段、生物材料(如DNA分子、表达载体、宿主细胞及其培养物)或包所述的抗Siglec15抗体或其抗原结合片段的药物组合物。
本发明所述的Siglec15介导的疾病或病症为表达Siglec15的癌症，如结直肠癌、子宫内膜样癌、甲状腺癌、膀胱癌、肾癌、肺癌、头颈部癌、乳腺癌、卵巢癌或肝癌。
本发明的抗Siglec15抗体及其抗原结合片段具有与Siglec15蛋白较高的结合活性，较好的体外逆转免疫细胞增殖抑制的功能，而且展现出更好的抑制肿瘤生长的效果。
附图说明
图1：本发明的鼠源抗体逆转hS15-Fc介导的对人T细胞抑制的结果。
图2：本发明的嵌合抗体逆转hS15-Fc介导的对人T细胞抑制的结果。
图3：本发明的嵌合抗体在MC38-humanSiglec15结直肠癌模型上的抗肿瘤活性。
图4：本发明的人源化抗体对于CHO-humanSiglec15细胞的结合能力。
图5：本发明的人源化抗体对于MC38-mouseSiglec15细胞的结合能力。
图6：本发明的IgG4型人源化抗体逆转hS15-Fc介导的对人T细胞抑制的结果，图中标示浓度表示抗体浓度。
图7：本发明的IgG1型人源化抗体逆转hS15-Fc介导的对人T细胞抑制的结果，图中标示浓度表示抗体浓度。
具体实施方式
术语和定义
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明所述的抗体是指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体的轻链恒定区。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、2、3、4或其变体的重链恒定区。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、 LCDR2和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)，或者符合kabat和chothia的编号规则。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的针对人Siglec15的单克隆抗体。制备时用Siglec15抗原注射试验对象(鼠)，然后分离表达具有所需序列或功能特性的抗体。在本发明一个优选的实施方案中，所述的鼠源Siglec15抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
在本发明一个优选的实施方案中，所述的抗Siglec15嵌合抗体的抗体轻链可变区进一步包含鼠源κ、λ链或其变体的轻链FR区，抗体轻链可变区序列如SEQ ID NO:8所示。所述的抗Siglec15嵌合抗体的抗体重链可变区进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区，抗体重链可变区序列如SEQ ID NO:7所示。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区。人抗体的轻链恒定区可选自人源κ、λ链或其变体的轻链恒定区，优选包含人源κ轻链恒定区。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指在不显著影响抗原结合特性的情况下，将非人来源(如小鼠)的CDR序列移植到人的抗体可变区框架。人源化抗体可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应的缺点。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时引起的活性下降，可对所述的人抗体可变区进行最少反向突变，以保持活性。
本发明中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段、Fab’片段、F(ab’) 2片段，以及与人Siglec15结合的Fv片段、scFv片段，包含本发明所述的SEQ ID NO:1至SEQ ID NO:6、SEQ ID NO:13或SEQ ID NO:14中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(scFv)。
Fab片段即由VL、VH、CL、CH1结构域组成的单价片段。
F(ab’)2即由通过铰链区处的二硫键连接的两个Fab片段的二价片段。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人Siglec15或其片段免疫，所得到的抗体能被复性，纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。
本发明所述的抗体或mAb，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术、合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种抗Siglec15抗体或其抗原结合片段的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻，如根据本领域已知的统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。在本发明中，所述抗体轻链或重链的变体即为在轻链或重链中发生0-10个氨基酸的“保守修饰”或“保守置换或取代”，本领域技术人员可以预期该变体具有与修饰或取代前基本相同的活性。
“有效量”包含足以改善或预防医学病症或症状的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指要根据背景在生物、细胞或人体外产生的物质。“内源性”指根据背景在细胞、生物或人体内产生的物质。
“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同一的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同一性。一般而言，当比对两个序列而得到最大的同一性百分率时进行比较。本领域技术人员可以判断序列同一性百分比所表示的变化的碱基数或氨基酸数量。
本文使用的表述“细胞”、“细胞系”和“细胞株”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同，包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“药物组合物”表示含有一种或多种本文所述抗Siglec15抗体或其抗原结合片段的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
在本发明中，Siglec15蛋白或S15蛋白或Siglec15抗原的GenBank登记号为NM_213602。本发明使用人Siglce15胞外区(20-263)作为抗原免疫小鼠，使其发生免疫反应产生针对Siglec15的鼠源抗体。
hS15-his是指在人Siglec15蛋白胞外区的C端连接6个组氨酸的融合蛋白。
hS15-Fc是指在人Siglec15蛋白胞外区的C端连接了人IgG1恒定区Fc片段的融合蛋白。
在本发明中，“hS15-Fc介导的对人T细胞的抑制”是指人Siglec15蛋白通过与免疫T细胞上的配体结合从而抑制免疫T细胞增殖的现象。
在本发明中，“抗人CD3抗体(克隆OKT3，biolegend)”是指针对免疫T细胞上的CD3抗原的抗体，用于激活T细胞的增殖。
MC38-humanSiglec15体内肿瘤模型为在C57BL/6N小鼠上建立的小鼠结直肠癌模型。将编码人Siglec15的cDNA序列(GenBank登记号NM_213602)转染至MC38空白细胞中，得到过表达人Siglec15的稳定细胞株MC38-humanSiglec15(MC38-hS15)。
本发明中涉及的编码抗Siglec15抗体的CDR、可变区或轻重链的DNA序列可以根据相应的氨基酸序列设计，这是本领域的常规技术。
以下结合实施例进一步描述本发明，但这些实施例并非限制着本发明的范围，本领域技术人员可由本说明书所公开的内容了解本发明的其他优点及效果。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原 料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1 Siglec15鼠源抗体的制备
1.1动物免疫
通过常规的动物免疫和融合方法制备针对人Siglec15的杂交瘤抗体，即Siglec15鼠源抗体：将编码人Siglec15的DNA插入表达载体，通过瞬转的方式转染Expi293F细胞(Lifetechnologies,12338-018)，进一步纯化得到免疫抗原人Siglec15。用500μg人Siglec15抗原与等体积的弗氏完全佐剂(Sigma-Aldrich)按照1:1混合乳化，乳液皮下免疫雌性C57BL/6N小鼠(北京维通利华)。随后，在第14天和28天皮下注射按照1:1混合的250μg人Siglec15蛋白和弗氏不完全佐剂(Sigma-Aldrich)的乳液，对小鼠进行加强免疫。第三次免疫完后，用酶联免疫吸附分析法(Elisa)测定小鼠血清的抗体滴度：将浓度为0.5μg/mL的hS15-his以100μL/孔的体积加入到96孔板中4℃包被过夜。用PBST(含有0.05％吐温20的磷酸盐缓冲液)洗涤3次后，并将其用200μL/孔的含1％牛血清蛋白(BSA)的PBST在37℃封闭1小时。随后弃去封闭液，向每个孔加入不同稀释比(首浓度用稀释液按照1：100稀释，后续按照2倍梯度稀释9个浓度)的100μL上述免疫的小鼠血清稀释液，然后在室温下孵育1小时。将板用PBST洗涤三次，并用100μL/孔的缀合辣根过氧化物酶的山羊抗小鼠IgG(Jackson Immunoresearch，115-035-003)在37℃孵育0.5小时。将板用PBST洗涤五次，然后加入TMB显色液(TIANGEN，S7717)并在室温下在黑暗中孵育15分钟。通过加入50μL的1mol/L HCl终止液(Sigma)终止反应，使用酶标仪在OD450nm的条件下读数并分析数据。在杂交瘤融合前4天，对展现出最高抗体滴度的2只小鼠进行Siglec15蛋白的加强免疫。
1.2杂交瘤融合和筛选
处死加强免疫的小鼠，提取脾脏并进行均质化以产生单细胞悬液，同时准备骨髓瘤细胞(SP2/0)(SGST)单细胞悬液。使用电融合将脾细胞与SP2/0小鼠骨髓瘤细胞进行融合。将融合的细胞重悬于含杂交瘤细胞选择剂DMEM+20％FBS+HAT(Gibco)的完全培养基中，并以200μL/孔接种至96孔板中。将96孔板在37℃ 5％CO 2的孵箱中培养6～7天，取细胞上清，采用ELISA检测鼠源抗体与Siglec15蛋白的结合情况。筛选出的优选杂交瘤克隆使用有限稀释法进行亚克隆。将杂交瘤细胞在37℃ 5％CO 2中培养1周后，对上清液进行ELISA结合检测，根据鼠源抗体与Siglec15蛋白结合及功能筛选出针对Siglec15的鼠源抗体E05。
对鼠源抗体E05可变区进行测序：提取杂交瘤细胞的mRNA，逆转录为cDNA后，通过通用引物进行PCR，将PCR得到的DNA产物进行测序分析，再翻译成氨基酸序列，并对可变区序列使用Kabat规则进行CDR区分析，其中，重链CDR1根据IMGT原则增加了5个氨基酸，得到结果如表1所示。
表1 Siglec15鼠源抗体E05的氨基酸序列
名称 序列编号
重链CDR1 SEQ ID NO:1
重链CDR2 SEQ ID NO:2
重链CDR3 SEQ ID NO:3
轻链CDR1 SEQ ID NO:4
轻链CDR2 SEQ ID NO:5
轻链CDR3 SEQ ID NO:6
重链可变区 SEQ ID NO:7
轻链可变区 SEQ ID NO:8
实施例2 Siglec15鼠源抗体逆转hS15-Fc介导的对人T细胞的抑制
抽取健康志愿者的外周血，利用密度梯度离心方法分离人外周血淋巴细胞(PBMC)，实验前将冻存的PBMC复苏并重悬于1640+10％FBS(Gibco)的完全培养基中；将抗人CD3抗体(克隆OKT3，biolegend，317302)用PBS稀释至0.05μg/mL，100μL/孔包被于96孔板中4℃过夜。实验前吸出未结合的OKT3上清液，并用PBS洗涤三遍；将可溶性人Siglec15与人Fc的融合蛋白(hS15-Fc)50μL/孔(终浓度1.5μg/ml)与E05抗体50μl/孔(2μg/ml)混合加入到96孔板中，同时设置不加E05(OKT3+hS15-Fc 1.5μg/ml)和不加hS15-Fc和E05抗体(OKT3)的对照孔；收集PBMC，用PBS洗涤一遍后用PBS重悬，加入5μM荧光染料CFSE(羟基荧光素二醋酸盐琥珀酰亚胺脂)在37℃下孵育15分钟，孵育完成后用PBS洗涤2次，用1640+10％FBS的完全培养基重悬至3E06的细胞密度，50μL/孔加入到96孔板中，96孔板在37℃、5％CO 2培养箱中孵育72小时。将细胞转移至圆底板96孔板上，用PBS缓冲液洗涤两次后，通过流式细胞术进行分析PBMC的增殖效果。
参照中国专利CN110035769A记载的方法制备得到鼠源抗体5G12(mIgG1)，作为阳性对照组，进行对照试验。
如图1所示：鼠源抗体E05可以显著的阻断hS15-Fc对T细胞的增殖抑制作用，且阻断效果优于阳性对照抗体5G12(图1中，“不加OKT3”为阴性对照，即只有PBMC细胞)。
实施例3 Siglec15嵌合抗体的制备
将鼠源抗体E05的重、轻链可变区分别定向插入含有信号肽和人IgG1恒定区、含有信号肽和人轻链kappa恒定区的表达载体pcDNA3.4(GenScript)中。
复苏Expi293F细胞至Expi293表达培养基(Gibco)中，在37℃ 8％CO 2的摇床中培养；转染前24小时将细胞密度调至2E6，转染前对细胞计数；将2ml预热的Opti-MEM(Gibco)培养基平分至两个离心管中，每管中加入相应量的抗体轻、重链质粒，并进行混匀；将100μl的转染试剂加入另一试管，室温孵育5分钟；将质粒与转染试剂混合，并室温孵育20分钟，然后加入到Expi293F细胞中；转染16小时后加入相应量的Enhancer1和Enhancer 2培养基 (Gibco)至细胞培养液中；培养表达5天后收集上清液，采用MabSelect PrismA(GE,10293703)进行纯化，得到Siglec15嵌合抗体ChE05。
表2 嵌合抗体ChE05的氨基酸序列
抗体名称 重链 轻链
ChE05 SEQ ID NO:9 SEQ ID NO:10
实施例4 Siglec15嵌合抗体逆转hS15-Fc介导的对人T细胞的抑制
参照实施例3的方法，同时参照中国专利CN110035769A制备得到人源化的5G12抗体(h5G12)作为阳性对照组进行对照试验，其中，CN110035769A仅公开h5G12的可变区，本申请发明人将其与人IgG1恒定区组合制备得到人源化的5G12抗体全长序列。
表2 对照抗体h5G12的氨基酸序列
抗体名称 重链 轻链
h5G12 SEQ ID NO:11 SEQ ID NO:12
参照实施例2的方法，检测本发明Siglec15嵌合抗体ChE05阻断hS15-Fc介导的对人T细胞的抑制。
如图2所示，Siglec15嵌合抗体ChE05可以较好的阻断hS15-Fc对T细胞的增殖抑制作用，且其阻断效果优于阳性对照抗体h5G12。
实施例5 Siglec15嵌合抗体体内抑制结直肠癌模型中肿瘤生长的效果
采用MC38-humanSiglec15体内肿瘤模型测量本发明抗体的体内免疫调节活性。
将编码人Siglec15的cDNA序列(GenBank登记号NM_213602)转染至MC38空白细胞中，得到过表达人Siglec15的稳定细胞株MC38-humanSiglec15(MC38-hS15)。C57BL/6N雌鼠(杭州医学院)，6～8周；收集MC38-hS15肿瘤细胞，PBS洗涤2遍后重悬至5E06的细胞密度，按照200μL/只的细胞量接种于C57BL/6N小鼠的右腹侧。接瘤后将小鼠随机分组，每组6只，接瘤数小时后开始腹腔给药，每只小鼠注射抗体10mg/kg，一周给药两次，共给药5次；每周3次测量小鼠体重和肿瘤大小，按(长度×宽度^2)*0.5计算肿瘤体积，每组小鼠取肿瘤体积的平均值作肿瘤生长曲线图。
采用实施例4中的h5G12抗体，作为阳性对照组，进行对照试验。
如图3显示，Siglec15嵌合抗体ChE05抑制MC38-hS15肿瘤生长的效果显著优于对照组h5G12。
实施例6 Siglec15人源化抗体的设计和抗体制备
在上述实施例获得鼠源抗体轻链可变区、重链可变区的基础上进行人源化改造。将鼠源抗体重链和轻链的6个CDR嫁接到与鼠源FR区具有较高相似度的人源模板上，人源模板是在PDB数据库通过BLAST获得与鼠源抗体序列相似度较高的germline序列，其中轻链可变区模 板为人种系轻链IGKV4-1*01，重链可变区的模板为人种系重链IGHV3-7*01。
CDR移植完成的抗体通过同源建模，预测在鼠抗FR区中可能决定抗体结构的关键氨基酸，此外对移植后的序列进行翻译后修饰(PTM)分析，发现抗体轻链可变区CDR1以及重链可变区的CDR2含有异构化修饰位点，采用回复突变的方法，将FR区个别人源模板的氨基酸回复突变回鼠源氨基酸，此外对CDR中PTM位点进行随机突变，通过亲和筛选，最终获得人源化的抗体。回复突变后的重链CDR2的氨基酸序列为SEQ ID NO:13，回复突变后的轻链CDR1的氨基酸序列为：SEQ ID NO:14。
对得到的人源化抗体进行测序：根据人源化抗体轻链可变区和重链可变区的氨基酸序列合成cDNA，并插入含有信号肽和人IgG1或IgG4(S228P)重链恒定区、含有信号肽和人轻链kappa恒定区的pcDNA3.4表达载体(GenScript)中，得到全长抗体的表达质粒。重链和轻链表达质粒共转染Expi293F细胞，培养6～7天后收获上清进行Protein A纯化，得到本发明的人源化抗体。
表3：人源化抗体序列
Figure PCTCN2022087405-appb-000001
实施例7 人源化抗体亲和力测定
采用Biacore T200生物大分子相互作用仪(GE Healthcare)进行人源化抗体亲和测定。在芯片上偶联抗人IgG-Fc抗体，利用抗人IgG抗体捕获Siglec15人源化抗体，以抗原Siglec15-his为流动相，使用6个浓度梯度(20、10、5、2.5、1.25、0.625、0.3125nM)，然后进行20秒缔合步骤和进行360秒的解离步骤。使用10mM甘氨酸-盐酸缓冲液进行再生，时间为60s；使用Biacore T200数据分析软件3.1处理数据。检测结果如表4所示。
表4：Biacore测定人源化抗体与Siglec15抗原的亲和力
Figure PCTCN2022087405-appb-000002
实验结果表明本发明的ChE05的人源化抗体都保持了和Siglec15结合活性，具有和嵌合抗体ChE05近似的亲和力。
实施例8 FACS测定Siglec15人源化抗体对于CHO-hS15和MC38-mS15细胞的结合
将编码人Siglec15和鼠Siglec15的cDNA序列(GenBank登记号NM_001101038.2)分别转染至CHO和MC38空白细胞中，得到过表达人Siglec15和鼠Siglec15的稳定细胞株CHO-HumanSiglec15(CHO-hS15)和MC38-mouseSiglec15(MC38-mS15)。将CHO-hS15和MC38-mS15用PBS重悬至细胞密度为4E06。将细胞按照50μL/孔加入到96孔板中，并添加50μL终浓度范围为30、10、3、1、0.3、0.1、0.03、0.01μg/ml的Siglec15人源化抗体。将细胞在4℃孵育30分钟，然后用PBS缓冲液洗涤两次。将细胞用100μL山羊抗人IgG-FITC二抗(Jackson Immunoresearch，109-545-003)1:200稀释液重悬，4℃孵育30分钟并用PBS缓冲液洗涤两次，通过流式细胞术进行分析，结果如图4、5所示。
结果表明，本发明的人源化抗体和ChE05嵌合抗体与CHO-hS15和MC38-mS15细胞的结合没有显著差别，且具有种属交叉性。
实施例9 Siglec15人源化抗体逆转hS15-Fc介导的对人T细胞的抑制
抽取健康志愿者的外周血，利用密度梯度离心方法分离人外周血淋巴细胞(PBMC)，实验前将冻存的PBMC复苏并重悬于1640+10％FBS的完全培养基中；将抗人CD3抗体(克隆OKT3，Biolegend)用PBS稀释至0.05μg/mL100μL/孔包被于在96孔板中4℃过夜。实验前吸出未结合的OKT3上清液，并用PBS洗涤三遍；将可溶性hS15-Fc 50μl/孔(终浓度1.5μg/ml)与不同浓度的本发明的S15人源化抗体(两种亚型IgG1和IgG4)50μl/孔(20、6.67、2.22、0.74、0.24μg/ml)混合加入到96孔板中，同时设置不加S15人源化抗体(OKT3+hS15-Fc 1.5μg/ml)和不加hS15-Fc和S15人源化抗体(OKT3)的对照孔；收集PBMC，用PBS洗涤一遍后用PBS重悬，加入5μM CFSE在37℃下孵育15分钟，孵育完成后用PBS洗涤2次，用1640+10％FBS的完全培养基重悬至3E06的细胞密度，50μL/孔加入到96孔板中，96孔板 在37℃CO 2培养箱中孵育72小时。将细胞转移至96孔圆底板上，用PBS缓冲液洗涤两次后，通过流式细胞术进行分析PBMC的增殖效果。
如图6、图7所示，本发明的Siglec15人源化抗体可以较好的阻断hS15-Fc对T细胞的增殖抑制作用，且阻断活性优于阳性对照h5G12抗体(图6和图7中，“不加OKT3”组为阴性对照；图6中“ChE05-4、h5G12-4”为将ChE05和h5G12的重链恒定区IgG1替换为IgG4所得)。
实施例10 Siglec15人源化抗体体内抑制肿瘤生长的效果
具体实施方法参照实施例5，采用MC38-humanSiglec15体内肿瘤模型测量本发明Siglec15人源化抗体的体内免疫调节活性，与不给药的空白组相比，本发明嵌合抗体和人源化抗体的抑制肿瘤的效果如表5所示。
表5：Siglec15人源化抗体体内抑制肿瘤生长的效果
组别 ChE05 E05-0 E05-1 E05-2 E05-5
抑瘤率％ 19.81 37.79 56.91 64.72 56.65
结果表明，本发明Siglec15人源化抗体与ChE05嵌合抗体相比，具有更加显著的抑制MC38-hSiglec15的肿瘤生长效果。抑瘤率＝((空白组体积-实验组体积)/空白组体积)*100％。
表6：序列信息
Figure PCTCN2022087405-appb-000003
Figure PCTCN2022087405-appb-000004
Figure PCTCN2022087405-appb-000005

Claims (14)

  1. 抗Siglec15抗体或其抗原结合片段，其包含：
    重链可变区，所述重链可变区包含至少1个如下HCDR：
    HCDR1，其氨基酸序列如SEQ ID NO:1所示，或包含SEQ ID NO:1所示氨基酸序列；
    HCDR2，其氨基酸序列如SEQ ID NO:2或SEQ ID NO:13所示，或包含SEQ ID NO:2或SEQ ID NO:13所示氨基酸序列；
    HCDR3，其氨基酸序列如SEQ ID NO:3所示，或包含SEQ ID NO:3所示氨基酸序列；和
    轻链可变区，所述轻链可变区包含至少1个如下LCDR：
    LCDR1，其氨基酸序列如SEQ ID NO:4或SEQ ID NO:14所示，或包含SEQ ID NO:4或SEQ ID NO:14所示氨基酸序列；
    LCDR2，其氨基酸序列如SEQ ID NO:5所示，或包含SEQ ID NO:5所示氨基酸序列；
    LCDR3，其氨基酸序列如SEQ ID NO:6所示，或包含SEQ ID NO:6所示氨基酸序列；
    优选地，所述的重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且所述的轻链可变区包含分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或者，
    所述的重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且所述的轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或者，
    所述的重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:13和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且所述的轻链可变区包含分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或者，
    所述的重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:13和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；且所述的轻链可变区包含分别如SEQ ID NO:14、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
  2. 如权利要求1所述的抗Siglec15抗体或其抗原结合片段，其中所述的重链可变区氨基酸序列如SEQ ID NO:7所示，或与SEQ ID NO:7具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，和/或所述的轻链可变区氨基酸序列如SEQ ID NO:8所示，或与SEQ ID NO:8具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
  3. 如权利要求1所述的抗Siglec15抗体或其抗原结合片段，为鼠源抗体或其抗原结合片段，进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区，和/或进一步包 含鼠源κ、λ链或其变体的轻链FR区；优选地，进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，和/或进一步包含鼠源κ、λ链或其变体的轻链恒定区。
  4. 如权利要求1所述的抗Siglec15抗体或其抗原结合片段，为嵌合抗体或其抗原结合片段，进一步包含人源κ、λ链或其变体的轻链恒定区，优选人源κ轻链恒定区；和/或进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区；
    优选地，所述抗体的重链氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；所述抗体的轻链氨基酸序列如SEQ ID NO:10所示，或与SEQ ID NO:10具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
  5. 如权利要求1所述的抗Siglec15抗体或其抗原结合片段，为人源化抗体或其抗原结合片段，进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区，优选包含人源IgG1或IgG4重链FR区；和/或进一步包含人源κ、λ链或其变体的轻链FR区，优选包含人源κ轻链FR区；
    优选地，其中所述的重链可变区氨基酸序列如SEQ ID NO:15、17、19或22所示，或与SEQ ID NO:15、17、19或22具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；和/或所述的轻链可变区氨基酸序列如SEQ ID NO:16、18、20或21所示，或与SEQ ID NO:16、18、20或21具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；
    优选地，所述抗Siglec15抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:15所示，或与SEQ ID NO:15具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区氨基酸序列如SEQ ID NO:16所示，或与SEQ ID NO:16具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；或者
    所述抗Siglec15抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:17所示，或与SEQ ID NO:17具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区氨基酸序列如SEQ ID NO:18所示，或与SEQ ID NO:18具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；或者
    所述抗Siglec15抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:19所示，或与SEQ ID NO:19具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区氨基酸序列如SEQ ID NO:20所示，或与SEQ ID NO:20具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；或者
    所述抗Siglec15抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:17所示，或与SEQ ID NO:17具有至少90％、至少95％、至少96％、至少97％、至少98％或至 少99％序列一致性，且其轻链可变区氨基酸序列如SEQ ID NO:20所示，或与SEQ ID NO:20具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；或者
    所述抗Siglec15抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:17所示，或与SEQ ID NO:17具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区氨基酸序列如SEQ ID NO:21所示，或与SEQ ID NO:21具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性；或者
    所述抗Siglec15抗体或其抗原结合片段的重链可变区氨基酸序列如SEQ ID NO:22所示，或与SEQ ID NO:22具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性，且其轻链可变区氨基酸序列如SEQ ID NO:20所示，或与SEQ ID NO:20具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性。
  6. 如权利要求5所述的抗Siglec15抗体或其抗原结合片段，进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1或IgG4重链恒定区；和/或进一步包含人源κ、λ链或其变体的轻链恒定区，优选人源κ轻链恒定区；
    优选地，所述的人源IgG1重链恒定区的氨基酸序列如SEQ ID NO：23所示，或所述的人源IgG4重链恒定区的氨基酸序列如SEQ ID NO：25所示，和/或所述人源κ轻链恒定区的氨基酸序列如SEQ ID NO：24所示。
  7. 如权利要求1-6任一项所述的抗Siglec15抗体或其抗原结合片段，其中所述的抗原结合片段选自Fab、Fv、scFv或F(ab’) 2。
  8. 一种生物材料，为：
    (1)编码如权利要求1-7任一项所述的抗体或其抗原结合片段的DNA分子；
    (2)含有如(1)所述的DNA分子的表达载体；
    (3)含有如(1)所述的DNA分子或如(2)所述的表达载体的宿主细胞或其培养物，其中所述的宿主细胞优选为人胚肾293细胞或中国仓鼠卵巢细胞。
  9. 一种生产抗Siglec15抗体或其抗原结合片段的方法，包括步骤：培养权利要求8所述的宿主细胞；优选地，还包括从获得的培养物中分离抗体；以及对所述抗体进行纯化。
  10. 一种药物组合物，其含有如权利要求1至7任一项所述的抗Siglec15抗体或其抗原结合片段和可药用的赋形剂、稀释剂或载体。
  11. 一种检测或诊断试剂盒，其含有权利要求1-7任一项所述的抗Siglec15抗体或其抗原结合片段。
  12. 如权利要求1-7任一项所述的抗Siglec15抗体或其抗原结合片段、如权利要求8所述的生物材料在制备药物中的用途，所述药物用于治疗或预防Siglec15介导的疾病或病症；优选地，所述的Siglec15介导的疾病或病症为表达Siglec15的癌症；进一步优选地，所述表达Siglec15的癌症为结直肠癌、子宫内膜样癌、甲状腺癌、膀胱癌、肾癌、肺癌、头颈部癌、乳腺癌、卵巢癌或肝癌。
  13. 一种治疗或预防Siglec15介导的疾病或病症的方法，包括给予所需患者治疗有效量的权利要求1-7任一项所述的抗Siglec15抗体或其抗原结合片段、权利要求8所述的生物材料、或权利要求10所述的药物组合物；优选地，所述的Siglec15介导的疾病或病症为表达Siglec15的癌症；进一步优选地，所述表达Siglec15的癌症为结直肠癌、子宫内膜样癌、甲状腺癌、膀胱癌、肾癌、肺癌、头颈部癌、乳腺癌、卵巢癌或肝癌。
  14. 如权利要求1-7任一项所述的抗Siglec15抗体或其抗原结合片段、如权利要求8所述的生物材料、如权利要求10所述的组合物或如权利要求11所述的试剂盒，在检测、诊断、预后Siglec15或Siglec15介导的疾病或病症中的用途；优选地，所述的Siglec15介导的疾病或病症为表达Siglec15的癌症；进一步优选地，所述表达Siglec15的癌症为结直肠癌、子宫内膜样癌、甲状腺癌、膀胱癌、肾癌、肺癌、头颈部癌、乳腺癌、卵巢癌或肝癌。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117447593B (zh) * 2022-07-26 2024-06-25 北京东方百泰生物科技股份有限公司 一种抗Siglec-15单克隆抗体

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110268733A1 (en) * 2009-04-09 2011-11-03 Daiichi Sankyo Company, Limited ANTI-Siglec-15 ANTIBODY
CN104321430A (zh) * 2012-03-30 2015-01-28 第一三共株式会社 新颖的抗-siglec-15抗体
CN104507969A (zh) * 2012-07-19 2015-04-08 阿莱斯亚生物疗法股份有限公司 抗siglec-15抗体
US20190202912A1 (en) * 2016-09-21 2019-07-04 Nextcure, Inc. Antibodies for siglec-15 and methods of use thereof
CN112159475A (zh) * 2020-10-10 2021-01-01 南京凯地生物科技有限公司 Siglec-15单克隆抗体及其应用
CN112694532A (zh) * 2021-01-12 2021-04-23 倍而达药业（苏州）有限公司 抗Siglec-15的抗体或其抗原结合片段及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1212334C (zh) * 2001-02-28 2005-07-27 第二军医大学免疫学研究所 人唾液酸结合性免疫球蛋白样凝集素，其编码序列及用途
US8168181B2 (en) * 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
US20170129956A1 (en) * 2014-06-18 2017-05-11 Daiichi Sankyo Company, Limited Anti-siglec-15 antibodies for use in treatment of osteogenesis imperfecta
US11104732B2 (en) * 2019-04-24 2021-08-31 Innovative Cellular Therapeutics Holdings, Ltd. Reducing immune inhibition induced by SIGLEC-15

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110268733A1 (en) * 2009-04-09 2011-11-03 Daiichi Sankyo Company, Limited ANTI-Siglec-15 ANTIBODY
CN104321430A (zh) * 2012-03-30 2015-01-28 第一三共株式会社 新颖的抗-siglec-15抗体
CN104507969A (zh) * 2012-07-19 2015-04-08 阿莱斯亚生物疗法股份有限公司 抗siglec-15抗体
US20190202912A1 (en) * 2016-09-21 2019-07-04 Nextcure, Inc. Antibodies for siglec-15 and methods of use thereof
CN112159475A (zh) * 2020-10-10 2021-01-01 南京凯地生物科技有限公司 Siglec-15单克隆抗体及其应用
CN112694532A (zh) * 2021-01-12 2021-04-23 倍而达药业（苏州）有限公司 抗Siglec-15的抗体或其抗原结合片段及应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

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