WO2022228183A1 - Anticorps anti-siglec 15, son procédé de préparation et son utilisation - Google Patents

Anticorps anti-siglec 15, son procédé de préparation et son utilisation 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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Chinese (zh)
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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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    • 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
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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
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    • 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
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    • 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

L'invention concerne un anticorps anti-Siglec 15, un fragment de liaison à l'antigène de celui-ci, l'utilisation médicale de celui-ci, un anticorps chimérique et un anticorps humanisé comprenant la région CDR de l'anticorps, une composition pharmaceutique comprenant l'anticorps anti-Siglec 15 et le fragment de liaison à l'antigène de celui-ci, ainsi que l'utilisation de l'anticorps dans la préparation d'un médicament pour le traitement de maladies ou d'affections.
PCT/CN2022/087405 2021-04-30 2022-04-18 Anticorps anti-siglec 15, son procédé de préparation et son utilisation WO2022228183A1 (fr)

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WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024040194A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo

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