WO2023030311A1 - Antigen-binding protein targeting siglec15 and use thereof - Google Patents

Antigen-binding protein targeting siglec15 and use thereof Download PDF

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Publication number
WO2023030311A1
WO2023030311A1 PCT/CN2022/115816 CN2022115816W WO2023030311A1 WO 2023030311 A1 WO2023030311 A1 WO 2023030311A1 CN 2022115816 W CN2022115816 W CN 2022115816W WO 2023030311 A1 WO2023030311 A1 WO 2023030311A1
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seq
amino acid
acid sequence
sequence shown
binding protein
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PCT/CN2022/115816
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French (fr)
Chinese (zh)
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廖雪梅
杨松霖
陈娜
王瓅
施伟军
栾珊珊
夏广新
柯樱
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上海医药集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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

Definitions

  • This application relates to the field of biomedicine, in particular to an antigen-binding protein targeting Siglec15 and its use.
  • Siglec15 is a type I transmembrane protein consisting of two Ig-like domains, a transmembrane domain containing lysine residues, and a short cytoplasmic tail. This protein was originally identified as a member of the Siglec gene family with a typical sialic acid-binding immunoglobulin-like lectin structure. Siglec15 mRNA is expressed at very low levels in most normal human tissues and in various immune cell subsets, but is higher in macrophages.
  • siglec15 is a macrophage-associated T cell inhibitory molecule, whose expression is usually limited to myeloid cells, but widely expressed in human tumor tissues. IFN- ⁇ can inhibit the expression level of Siglec15, and Siglec15 inhibits T cell activity in vitro.
  • the physiological indicators of Siglec15 knockout mice were normal, showing the safety of the Siglec15 target.
  • Siglec15 inhibits antigen-specific T cell responses by inhibiting IL-10.
  • Siglec15 knockout mice increase the secretion of CD8+T cells, NK cells and cytokines, reduce the incidence of tumors, and increase the survival rate. The above results suggest that Siglec15 may be a very potential target for tumor therapy.
  • the antigen-binding proteins targeting Siglec15 still have the defects of poor selectivity and low affinity. Therefore, it is necessary to develop novel antigen-binding proteins with high affinity and strong specificity targeting Siglec15.
  • the application provides an isolated antigen-binding protein, which has one or more of the following properties: a) specifically binds to human Siglec15 protein with a KD value of about 5E-09M or below in a Biacore assay ; b) in flow cytometry, it specifically binds to human Siglec15 expressed on the surface of CHOK1 cells with an EC50 value of about 0.2 ⁇ g/ml or below; c) releases Siglec15 from inhibiting the proliferation of PBMCs in blood; d) detects in ELISA Among them, it specifically binds to human Siglec15 with an EC50 value of about 0.2 ⁇ g/ml or below; e) In ELISA detection, it specifically binds to monkey Siglec15 with an EC50 value of about 0.1 ⁇ g/ml or below; f) In ELISA detection wherein, specifically binds to murine Siglec15 with an EC50 value of about 0.1 ⁇ g/ml or less; and
  • the isolated antigen-binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:1.
  • the isolated antigen-binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.
  • the isolated antigen-binding protein comprises HCDR1 comprising the amino acid sequence [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein , X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S.
  • the HCDR1 in the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:20 or SEQ ID NO:22.
  • said isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA Amino acid sequence, wherein X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22, wherein Said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the isolated antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 29.
  • the H-FR1 in the isolated antigen binding protein comprises SEQ ID NO:9 or the amino acid sequence shown in SEQ ID NO:9.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:32 amino acid sequence.
  • the H-FR2 in the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:31 amino acid sequence.
  • said H-FR3 in said isolated antigen binding protein comprises SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: The amino acid sequence shown in 27.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 33.
  • the H-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  • the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 29, wherein The H-FR2 comprises the amino acid sequence shown in SEQ ID NO:32, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:33 .
  • said isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NO:4 or SEQ ID NO:9
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:5
  • the H-FR3 comprises SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO: 14.
  • the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:27, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups: 1) the H-FR1 comprises SEQ ID NO The amino acid sequence shown in: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 6 The amino acid sequence shown in ID NO:7; 2) said H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, and said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said H- FR3 comprises the amino acid sequence shown in SEQ ID NO:11, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 3) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, The H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, the H-FR3 comprises the amino acid sequence shown in SEQ ID
  • the isolated antigen-binding protein comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:34.
  • said isolated antigen binding protein comprises a heavy chain variable region VH comprising SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16 , SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:28 amino acid sequence.
  • the isolated antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region.
  • said heavy chain constant region of said isolated antigen binding protein comprises a constant region derived from human IgG.
  • the heavy chain constant region in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38 .
  • the isolated antigen binding protein comprises an antibody or antigen binding fragment.
  • the antigen-binding fragment of the isolated antigen-binding protein is selected from the group consisting of: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • said antibody in said isolated antigen binding protein is selected from the group consisting of monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.
  • the isolated antigen binding protein is a single domain antibody.
  • the single domain antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:1.
  • the single domain antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.
  • the single domain antibody comprises HCDR1
  • the HCDR1 comprises the amino acid sequence represented by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein, X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, and X 5 is S.
  • the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22.
  • the single domain antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence represented by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, Wherein, X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the single domain antibody comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22, and the HCDR2 comprises The amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1.
  • the single domain antibody comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises SEQ ID NO:29 The amino acid sequence shown.
  • the single domain antibody comprises H-FR1, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:9.
  • the single domain antibody comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:32 .
  • the single domain antibody comprises H-FR2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:9.
  • the single domain antibody comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31 .
  • the single domain antibody comprises H-FR3 comprising amino acids shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:18 sequence.
  • the single domain antibody comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO:33 The amino acid sequence shown.
  • the single domain antibody comprises H-FR4 comprising the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  • the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 29, the H- FR2 comprises the amino acid sequence shown in SEQ ID NO:32, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises amino acids shown in SEQ ID NO:4 or SEQ ID NO:9 Sequence
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10
  • the H-FR3 comprises SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:14 or SEQ ID NO:10
  • the amino acid sequence shown in ID NO:18, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  • the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups; 1) the H-FR1 comprises SEQ ID NO:4 The amino acid sequence shown, the H-FR2 includes the amino acid sequence shown in SEQ ID NO:5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 7; 2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:10 The amino acid sequence shown in ID NO:11, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 3) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the H -FR2 comprises the amino acid sequence shown in SEQ ID NO:10, the H-FR3 comprises the
  • the single domain antibody comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:34.
  • the single domain antibody comprises a heavy chain variable region VH comprising SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID The amino acid sequence shown in NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:28 .
  • the single domain antibody comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region.
  • said heavy chain constant region of said single domain antibody comprises a constant region derived from human IgG.
  • the heavy chain constant region of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.
  • the present application provides a polypeptide comprising the isolated antigen-binding protein described herein.
  • the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein or the polypeptide described herein.
  • the present application provides an isolated nucleic acid molecule encoding the isolated antigen-binding protein described herein, or the polypeptide described herein.
  • the present application provides a vector comprising the isolated nucleic acid molecule described herein.
  • a cell comprising the isolated antigen binding protein described in the present application, the polypeptide described in the present application, the immunoconjugate described in the present application, the isolated nucleic acid described in the present application Molecules and/or vectors described herein.
  • the application provides a method for preparing the isolated antigen-binding protein described in the application or the polypeptide described in the application, the method comprising making the isolated antigen-binding protein described in the application or the polypeptide described in the application.
  • the cells described in the present application are cultured under the condition that the polypeptide is expressed.
  • the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein described in the present application, the polypeptide described in the present application, the immunoconjugate described in the present application, the isolated antigen-binding protein described in the present application
  • the present application provides a pharmaceutical combination comprising the isolated antigen-binding protein described in the present application and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor includes a substance that inhibits PD-1/PD-L1 interaction.
  • the immune checkpoint inhibitor is selected from the group consisting of PD-1/PD-L1 blockers, PD-1 antagonists, PD-L1 antagonists, PD-1 inhibitors, and PD- L1 inhibitor.
  • the immune checkpoint inhibitor comprises an anti-PD-1 antibody.
  • the anti-PD-1 antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:39.
  • the anti-PD-1 antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:40.
  • the anti-PD-1 antibody comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41.
  • the anti-PD-1 antibody comprises a heavy chain variable region VH
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 39
  • the HCDR2 Comprising the amino acid sequence shown in SEQ ID NO:40
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:41.
  • the anti-PD-1 antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 42.
  • the anti-PD-1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:43.
  • the anti-PD-1 antibody comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO:44.
  • the anti-PD-1 antibody comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:45.
  • the anti-PD-1 antibody comprises a light chain variable region VL
  • the VL comprises LCDR1, LCDR2 and LCDR3
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 43
  • the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:44
  • the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:45.
  • the anti-PD-1 antibody comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:46.
  • the anti-PD-1 antibody comprises pembrolizumab.
  • the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:47.
  • the anti-PD-L1 antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:48.
  • the anti-PD-L1 antibody comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:49.
  • the anti-PD-L1 antibody comprises a heavy chain variable region VH
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 47
  • the HCDR2 Comprising the amino acid sequence shown in SEQ ID NO:48
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:49.
  • the anti-PD-L1 antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:50.
  • the anti-PD-L1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:51.
  • the anti-PD-L1 antibody comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:52.
  • the anti-PD-L1 antibody comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53.
  • the anti-PD-L1 antibody comprises a light chain variable region VL
  • the VL comprises LCDR1, LCDR2 and LCDR3
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:52
  • the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53.
  • the anti-PD-L1 antibody comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:54.
  • the anti-PD-L1 antibody comprises atezolizumab.
  • the present application provides a method for detecting or measuring Siglec15, the method comprising using the isolated antigen-binding protein described in the present application or the polypeptide described in the present application.
  • the present application provides a Siglec15 detection kit, which comprises the isolated antigen-binding protein or the polypeptide described in the present application.
  • the present application provides a method for regulating immune response, which comprises administering to a subject in need an effective amount of the isolated antigen-binding protein described in the present application, the polypeptide described in the present application, the polypeptide described in the present application, The immunoconjugate described herein, the isolated nucleic acid molecule described herein, the carrier described herein, the cell described herein, the pharmaceutical composition described herein and/or the pharmaceutical combination described herein, and/or a pharmaceutically acceptable therapeutic agent.
  • the application provides an isolated antigen-binding protein described in the application, the polypeptide described in the application, the immunoconjugate described in the application, the isolated nucleic acid molecule described in the application, the polypeptide described in the application Use of the carrier, the cell described in the application, the pharmaceutical composition described in the application and/or the combination of drugs described in the application in the preparation of drugs for preventing and/or treating diseases or diseases.
  • the disease or disorder in the use of the medicament for preventing and/or treating a disease or disorder described in the present application, includes a tumor.
  • the tumor in the application of the medicament for preventing and/or treating diseases or conditions described in this application, includes a tumor associated with the expression of Siglec15.
  • the tumor in the use of the medicament for preventing and/or treating a disease or condition described in this application, includes a solid tumor.
  • the tumor in the use of the medicament for preventing and/or treating diseases or conditions described in this application, includes colon cancer.
  • Figure 1 shows the binding curve of the Siglec15-binding protein described in the present application to human Siglec15 expressed on the surface of CHOK1 cells.
  • FIG. 2A shows that the Siglec15 antigen-binding protein described in this application promotes the proliferation of CD8 + T cells.
  • FIG. 2B shows that the Siglec15 antigen-binding protein described in this application promotes the proliferation of CD4 + T cells.
  • FIG. 2C shows that the Siglec15 antigen-binding protein described in this application promotes the expression of IFN- ⁇ in the culture supernatant.
  • 3A-B show the binding curves of the humanized Siglec15-binding protein described in the present application and human Siglec15 expressed on the surface of CHOK1 cells.
  • FIG. 4A shows that the humanized Siglec15 antigen-binding protein described in this application promotes the proliferation of CD8 + T cells.
  • Figure 4B shows that the humanized Siglec15 antigen-binding protein described in this application promotes the proliferation of CD4 + T cells.
  • Figure 4C shows that the humanized Siglec15 antigen-binding protein of the present application promotes the expression of IFN- ⁇ in the culture supernatant.
  • 5A-B show the binding curves of the humanized Siglec15 antigen-binding protein described in the present application to human Siglec15 antigen.
  • 5C-D show the binding curves of the humanized Siglec15 antigen-binding protein described in the present application to the monkey Siglec15 antigen.
  • 5E-F show the binding curves of the humanized Siglec15 antigen-binding protein described in the present application to the mouse Siglec15 antigen.
  • Figure 6A shows the anti-tumor efficacy of the antigen-binding protein described in this application in wild-type C57BL/6N mice.
  • Fig. 6B shows the body weight changes of wild-type C57BL/6N mice after administration of the antigen-binding protein described in the present application.
  • Figure 7A shows the anti-tumor efficacy of the antigen-binding protein described in this application in human Siglec15 transgenic mice.
  • Fig. 7B shows the body weight changes of human Siglec15 transgenic mice after administration of the antigen-binding protein described in the present application.
  • Figure 8A shows the anti-tumor efficacy of the antigen-binding protein described in this application in human Siglec15 transgenic mice.
  • Fig. 8B shows the body weight changes of human Siglec15 transgenic mice after administration of the antigen-binding protein described in the present application.
  • Figure 9 shows that the humanized Siglec15 antigen-binding protein described in the present application inhibits the formation of osteoclasts.
  • Figure 10A shows the anti-tumor efficacy of the combination of antigen-binding proteins Ab5, Ab10 and PD-1 antibody described in this application in human PD-1/PD-L1 transgenic mice.
  • Figure 10B shows the body weight changes of human PD-1/PD-L1 transgenic mice after the combination of antigen binding proteins Ab5, Ab10 and PD-1 antibody described in this application.
  • Figure 11A shows the anti-tumor efficacy of the combined use of antigen-binding proteins Ab5, Ab10 and PD-L1 antibodies described in this application in human PD-1/PD-L1 transgenic mice.
  • Figure 11B shows the body weight changes of human PD-1/PD-L1 transgenic mice after the combination of antigen binding proteins Ab5, Ab10 and PD-L1 antibody described in this application.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • the terms "Siglec15 protein”, “Siglec-15” or “Siglec15 antigen” are used interchangeably and include any functionally active fragments, variants and homologues of Siglec15 that are naturally expressed by cells or used in Expression on Siglec15 gene transfected cells.
  • the Siglec15 described in the present application may include Siglec15 antigen or a fragment thereof or an antigenic determinant thereof.
  • the Siglec15 described in the present application may include human Siglec15, mouse Siglec15 and/or monkey Siglec15.
  • the human Siglec15 is human Siglec15.
  • the nucleotide sequence number encoding it may be 284266, and its accession number in UniProt/Swiss-Prot may be Q6ZMC9.
  • the mouse Siglec15 is mouse Siglec15, and the nucleotide sequence code encoding it may be 620235 in NCBI.
  • the monkey Siglec15 is cynomolgus monkey Siglec15, and the nucleotide sequence code encoding it may be 700656 in NCBI.
  • the term "functionally active fragment” refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence.
  • a functionally active fragment of any given sequence is one in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least A sequence of endogenous function.
  • Sequences encoding functionally active fragments can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in naturally occurring proteins and/or polynucleotides , as long as the original functional activity is maintained.
  • derivative generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and and/or added, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analogue generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.
  • amino acid substitutions e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.
  • homologue generally refers to an amino acid sequence or nucleotide sequence having a certain homology to a naturally occurring sequence.
  • the term “homology” may be equated with sequence "identity”.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will comprise the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity.
  • sequence having a percentage identity of any one of the SEQ ID NOs of the mentioned amino acid sequence or nucleotide sequence means having said percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.
  • sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.
  • proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid.
  • Paragine, Glutamine, Serine, Threonine and Tyrosine are examples of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • the term "antigen-binding protein” generally refers to an antibody or fragment thereof capable of binding to an antigen or fragment thereof.
  • the antigen binding protein may include humanized antibody, chimeric antibody, recombinant antibody, single chain antibody, monoclonal antibody, polyclonal antibody, Fab fragment, F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody , IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and/or IgG4 antibodies, and fragments thereof.
  • the antigen-binding proteins described in the present application may include antigen-binding proteins derived from humans, mice, monkeys and/or alpacas.
  • the antigen binding protein described in the present application may comprise a humanized Siglec15 single domain antibody.
  • the term "specific binding” generally refers to an interaction in which a binding protein (such as an antigen-binding protein) specifically recognizes a ligand (such as an antigen) and binds to it.
  • the specific binding described in the present application may include at least one species-derived antigen-binding protein, antigen-binding protein fragment and/or polypeptide and at least one species-derived antigen, antigenic determinant, antigenic epitope, due to a specific spatial conformation Identify and combine.
  • the specific binding described in the present application may include that the antigen binding protein described in the present application specifically recognizes Siglec15 and blocks the function of Siglec15.
  • K D (likewise, “K D " or “K D ”) generally refers to "affinity constant” or "equilibrium dissociation constant” and refers to a titration measurement at equilibrium, or Value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka). Binding of a binding protein (e.g., an isolated antigen-binding protein described herein) to an antigen (e.g., Siglec15 protein) is expressed using an on-rate constant (ka), an off-rate constant (kd), and an equilibrium dissociation constant ( KD ) affinity. Methods for determining association and dissociation rate constants are well known in the art.
  • the KD value can be determined by Biacore (Biomolecular Interaction Analysis) (for example, an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden), and other experimental approaches and instruments such as Octet can also be used.
  • the KD value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho), or using a surface plasmon resonance (SPR) instrument.
  • the KD value can also be determined by an amine coupling kit.
  • proliferation inhibition generally refers to the process of arresting or slowing cell division (symmetric or asymmetric division of cells) or cell growth.
  • the “proliferation inhibition” mentioned in this application may mean that Siglec15 can inhibit the activation, growth and/or division of T cells in the tumor microenvironment.
  • the term "antibody” generally refers to a class of immunoglobulins that can specifically bind to a specific antigen, protein, fragment or polypeptide and exert functions.
  • the antibody may comprise: a heavy chain constant region, a light chain constant region, a heavy chain variable region and/or a light chain variable region.
  • the variable region may comprise separate CDR regions and separate FR regions.
  • the constant region may comprise an isolated CH1 domain, an isolated CH2 domain and/or an isolated CH3 domain.
  • Antibodies described herein may include whole antibodies, any binding fragments and/or single chains thereof.
  • any of the binding fragments may include: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibodies described herein may include monoclonal antibodies, polyclonal antibodies, single chain antibodies, single domain antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
  • the antibodies described herein may include antibodies derived from humans, mice, monkeys and/or alpacas.
  • the term "monoclonal antibody” generally refers to a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
  • Monoclonal antibodies are highly specific, directed against a single antigenic site.
  • the monoclonal antibodies can be produced by hybridoma technology or produced in bacterial, eukaryotic or plant cells by using recombinant DNA methods.
  • Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991) conduct.
  • chimeric antibody generally refers to an antibody in which a part of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class, and The remainder of the chain is then homologous to the corresponding sequence in another species.
  • the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human) .
  • B cells or hybridoma cells of non-human origin can be used to produce variable regions combined with constant regions of human origin.
  • variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region it is combined with.
  • the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody whose constant region is of non-human origin.
  • humanized antibody generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing the immunogenicity of a heterologous antibody when introduced into a human, while simultaneously Preserves the full antigen-binding affinity and specificity of the antibody.
  • CDR grafting Jones et al., Nature 321:522 (1986) and variants thereof; including “reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), "high addition” (hyperchimerization), (Queen, et al., 1989Proc Natl Acad Sci USA 86 : 10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co, et al., 1992J Immunol 148: 1149-1154) and "veneering” (veneering), (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.”
  • Metcalf B W Dalton B J
  • Dalton B J Da
  • single domain antibody generally refers to Nanobodies that are naturally devoid of light chains.
  • the single domain antibody described in this application may include heavy chain variable regions and conventional heavy chain CH2 and CH3 regions.
  • the single domain antibody described in the present application can be the smallest binding unit of an antigen binding protein.
  • the single domain antibody described herein may comprise Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the term "antigen-binding fragment” generally refers to one or more fragments of an antibody that specifically bind to an antigen.
  • the antigen-binding function of antibodies can be realized by full-length fragments of antibodies.
  • the antigen-binding function of an antibody can also be achieved by comprising a heavy chain of a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab'), or by comprising a Fv, scFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2.
  • Fab fragment usually a monovalent fragment consisting of VL, VH, CL and CH domains;
  • F(ab')2 fragment comprising two Fab fragments linked by a disulfide bond at the hinge region (3) Fd fragment composed of VH and CH domains; (4) Fv fragment composed of VL and VH domains of antibody single arm; (5) dAb fragment composed of VH domains (Ward et al., (1989) Nature 341 :544-546); (6) an isolated complementarity determining region (CDR) and (7) a combination of two or more isolated CDRs optionally linked by a linker.
  • CDR complementarity determining region
  • the monovalent single-chain molecule Fv formed by the pairing of VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl.Acad.Sci.85 :5879-5883).
  • a type of antibody VHH that lacks the light chain of the antibody and only has the variable region of the heavy chain can also be included (for example, see Kang Xiaozhen et al., Acta Biological Engineering, 2018, 34(12): 1974-1984).
  • the "antigen binding portion” may also include an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • heavy chain constant region generally refers to an isolated CH1 domain, an isolated CH2 domain and/or an isolated CH3 domain in an immunoglobulin heavy chain structure.
  • the heavy chain constant region described in the present application may include a fragment connected to the C-terminus of the VH fragment of the heavy chain variable region in the structure of the single domain antibody.
  • the heavy chain constant region described herein may comprise an IgG-derived constant region.
  • the term "heavy chain variable region” or “VH” generally refers to the region of the immunoglobulin heavy chain structure comprising the heavy chain complementarity determining regions CDR1, CDR2, CDR3 and the framework regions FR1, FR2, FR3, FR4 .
  • the heavy chain variable region contains a binding domain that interacts with the antigen.
  • the heavy chain variable region described in the present application may include a region that specifically binds to Siglec15 in the single domain antibody structure.
  • the heavy chain variable region described herein may comprise the VHH of a single domain antibody.
  • the term "encoding” generally refers to the property of a nucleic acid (such as deoxyribonucleic acid) transcribing a complementary nucleic acid, including nucleic acid capable of being translated into a polypeptide.
  • deoxyribonucleic acid can encode RNA transcribed from deoxyribonucleic acid.
  • a deoxyribonucleic acid may similarly encode a polypeptide translated from RNA transcribed from the deoxyribonucleic acid.
  • IgG generally refers to a class of polypeptides of the antibody class encoded by immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3 and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b, and IgG3.
  • the IgG described herein includes a heavy chain constant region, a light chain constant region, a heavy chain variable region and/or a light chain variable region.
  • the IgG constant region described herein can provide a heavy chain constant region structure for a humanized single domain antibody.
  • immunoconjugate generally refers to other components linked to an antigen-binding protein or fragment thereof.
  • the immunoconjugates may include other polypeptides, therapeutic agents, probes and/or other antibodies fused or conjugated to the antigen binding protein.
  • nucleic acid molecule generally refers to a biomacromolecular compound composed of many nucleotide monomers, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • the nucleic acid molecule can be single-stranded or double-stranded.
  • the nucleic acid molecule described in the present application may be any nucleic acid molecule encoding the antibody or peptide involved in the present application.
  • the nucleic acid molecules described herein may be present in cells, in vectors and/or in free form in water or solutions.
  • the nucleic acid molecules may include naturally occurring or synthetic nucleic acid molecules.
  • the term "vector” generally refers to a self-replicating DNA molecule that transfers DNA fragments to recipient cells in genetic engineering recombinant DNA technology.
  • the vectors may include bacterial plasmid vectors, phage vectors and/or animal and plant viral vectors.
  • the vector described in this application may be a circular DNA vector, including an origin of replication, a multiple cloning site, an open reading frame and/or a marker gene.
  • amino acid sequence generally refers to the sequence formed by linking at least one amino acid to each other.
  • the amino acid sequence may encode the main structure of a polypeptide or protein. Amino acids in the amino acid sequence can be linked in the form of dehydration condensation.
  • the amino acid sequences described herein may encode various antigen binding proteins or fragments thereof.
  • polypeptide can be used interchangeably, and generally refer to a compound composed of one or more amino acids linked together.
  • the polypeptide may include a compound formed by dehydration condensation of at least one amino acid with other amino acids.
  • Polypeptides described herein may include antigen binding proteins and fragments thereof.
  • the polypeptides described herein may include natural amino acid polymers and artificially synthesized amino acid polymers.
  • the term "cell” generally refers to an individual cell that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing a polypeptide described herein or an antigen binding protein described herein , cell line or cell culture.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the polypeptide or antigen-binding protein described in this application.
  • the cells can be obtained by transfecting cells in vitro with the vectors described in this application.
  • the cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • the cells can be immune cells.
  • the immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.
  • detection kit generally refers to a set of chemical reagents and/or biomacromolecules for detection of proteins and/or nucleic acid molecules.
  • the term “immunoconjugate” generally refers to a conjugate formed by conjugating (for example, covalently linking through a linker molecule) the other therapeutic agent to the isolated antigen-binding protein, the conjugate
  • the other therapeutic agent can be delivered to a target cell (eg, a tumor cell) by specific binding of the isolated antigen binding protein to an antigen on the target cell.
  • the antigen may also be secreted by the target cell and located in the space outside the target cell.
  • the term "pharmaceutical composition” generally refers to a composition comprising one or more biological activities of a drug.
  • the pharmaceutical composition described in the application can comprise the antigen-binding protein described in the application, the nucleic acid molecule described in the application, the vector described in the application and/or the cell described in the application, and optionally pharmaceutically acceptable Accepted adjuvants.
  • the term "tumor” generally refers to a neoplasm formed by the proliferation of local tissue cells.
  • the tumor can include a solid tumor.
  • the tumor can include a tumor associated with expression of Siglec15.
  • the term "tumor associated with expression of Siglec15” generally refers to a tumor in which Siglec15 expression is present.
  • the tumor associated with Siglec15 protein expression may be a Siglec15 positive tumor.
  • the protein expression of Siglec15 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • solid tumor generally refers to a tangible mass that can be detected clinically (eg, X-ray film, CT scan, B-ultrasound or palpation).
  • the solid tumor can include colon cancer.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen binding protein.
  • the isolated antigen-binding protein may have a KD value of about 5E-09M or less in a Biacore assay (for example, the KD value is not higher than about 5E-09M, not higher than about 4.9E-09M, Not higher than about 4.8E-09M, not higher than about 4.7E-09M, not higher than about 4.6E-09M, not higher than about 4.5E-09M, not higher than about 4.4E-09M, not higher than about 4.3 E-09M, not higher than about 4.2E-09M, not higher than about 4.1E-09M, not higher than about 4E-09M, not higher than about 3.0E-09M, not higher than about 2.5E-09M, not higher At about 2.0E-09M, not higher than about 1.5E-09M, not higher than about 1.0E-09M, not higher than about 9E-10M, not higher than about 8E-10M, not higher than about 5E-11M, not higher
  • the isolated antigen-binding protein may have an EC 50 value of about 0.2 ⁇ g/ml or less in flow cytometry (for example, the EC 50 value is not higher than about 0.2 ⁇ g/ml, not More than about 0.15 ⁇ g/ml, not more than about 0.13 ⁇ g/ml, not more than about 0.12 ⁇ g/ml, not more than about 0.1 ⁇ g/ml, not more than about 0.09 ⁇ g/ml, not more than about 0.08 ⁇ g /ml, not higher than about 0.07 ⁇ g/ml, not higher than about 0.06 ⁇ g/ml, not higher than about 0.05 ⁇ g/ml, not higher than about 0.04 ⁇ g/ml, not higher than about 0.03 ⁇ g/ml, not higher at about 0.02 ⁇ g/ml or not higher than about 0.01 ⁇ g/ml or lower) specifically binds to human Siglec15 expressed on the surface of CHOK1 cells.
  • the EC 50 value
  • the isolated antigen-binding protein may have an EC 50 value of about 0.2 ⁇ g/ml or less in ELISA detection (for example, the EC 50 value is not higher than about 0.2 ⁇ g/ml, not higher At about 0.19 ⁇ g/ml, not higher than about 0.18 ⁇ g/ml, not higher than about 0.17 ⁇ g/ml, not higher than about 0.16 ⁇ g/ml, not higher than about 0.15 ⁇ g/ml, not higher than about 0.1 ⁇ g/ml ml, not more than about 0.09 ⁇ g/ml, not more than about 0.08 ⁇ g/ml, not more than about 0.07 ⁇ g/ml, not more than about 0.06 ⁇ g/ml, not more than about 0.05 ⁇ g/ml or not more than About 0.01 ⁇ g/ml or less) specifically binds to human Siglec15.
  • the isolated antigen-binding protein may have an EC 50 value of about 0.1 ⁇ g/ml or less in ELISA detection (for example, the EC 50 value is not higher than about 0.1 ⁇ g/ml, not higher At about 0.09 ⁇ g/ml, not higher than about 0.08 ⁇ g/ml, not higher than about 0.07 ⁇ g/ml, not higher than about 0.06 ⁇ g/ml, not higher than about 0.05 ⁇ g/ml, not higher than about 0.04 ⁇ g/ml ml, not higher than about 0.03 ⁇ g/ml, not higher than about 0.02 ⁇ g/ml, or not higher than about 0.01 ⁇ g/ml or less) specifically binds to monkey Siglec15.
  • the EC 50 value is not higher than about 0.1 ⁇ g/ml, not higher At about 0.09 ⁇ g/ml, not higher than about 0.08 ⁇ g/ml, not higher than about 0.07 ⁇ g/ml, not higher than about 0.
  • the isolated antigen-binding protein may have an EC 50 value of about 0.1 ⁇ g/ml or less in ELISA detection (for example, the EC 50 value is not higher than about 0.1 ⁇ g/ml, not higher At about 0.09 ⁇ g/ml, not higher than about 0.08 ⁇ g/ml, not higher than about 0.07 ⁇ g/ml, not higher than about 0.06 ⁇ g/ml, not higher than about 0.05 ⁇ g/ml, not higher than about 0.04 ⁇ g/ml ml, not higher than about 0.03 ⁇ g/ml, not higher than about 0.02 ⁇ g/ml, or not higher than about 0.01 ⁇ g/ml or less) specifically binds to murine Siglec15.
  • the EC 50 value is not higher than about 0.1 ⁇ g/ml, not higher At about 0.09 ⁇ g/ml, not higher than about 0.08 ⁇ g/ml, not higher than about 0.07 ⁇ g/ml, not higher than about
  • the isolated antigen-binding protein can relieve Siglec15 from inhibiting the proliferation of PBMC in blood.
  • releasing the inhibition of Siglec15 on the proliferation of PBMCs in blood may include releasing the inhibition of Siglec15 on the proliferation of T cells.
  • the release of Siglec15's inhibition of T cell proliferation may include promoting the proliferation of CD8 + and CD4 + T cells and/or promoting the expression of IFN- ⁇ .
  • the isolated antigen binding protein inhibits the growth and/or proliferation of tumor cells.
  • the inhibition of the growth and/or proliferation of tumor cells includes that in the tumor microenvironment, the isolated antigen-binding protein releases the inhibition of Siglec15 on immune cells by targeting the Siglec15 on the surface of immune cells, thereby activating Inhibition of tumor cell growth and/or proliferation by immune cells.
  • the application provides an isolated antigen binding protein.
  • the isolated antigen binding protein may comprise an antibody or an antigen binding fragment.
  • the antigen-binding fragments may include: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibodies may include: monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
  • the isolated antigen-binding protein may be a single domain antibody.
  • the application provides an isolated antigen-binding protein, which may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the VH can comprise SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: The amino acid sequence shown in any one of ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:28.
  • the HCDR of the isolated antigen-binding protein can be divided by any form, as long as VH is compatible with SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO :17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:28 shown in any one
  • the amino acid sequences of the HCDRs are the same, and the HCDRs obtained by dividing them in any form can fall within the protection scope of the present application.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the isolated antigen binding proteins described herein are defined by the IMGT coding system.
  • the antigen-binding protein may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the HCDR3 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the HCDR2 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein , X1 can be G or R, X2 can be F or S, X3 can be I or T, X4 can be F, X5 can be S.
  • the HCDR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • HCDR1 of the antigen binding protein has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 ,X 3 ,X 4 ,X 5 .
  • X 1 can be G or R
  • X 2 can be F or S
  • X 3 can be Is I or T
  • X 4 can be F
  • X 5 can be S.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:22.
  • the HCDR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA Amino acid sequence, wherein, X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S, and the HCDR2 may comprise the amino acid shown in SEQ ID NO:2 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22.
  • the antigen binding protein may comprise antibodies Ab0 to Ab11 or an antigen binding fragment having the same HCDRs (eg, having the same HCDR1-3) therewith.
  • the VH of the antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3 and/or H-FR4.
  • the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO : the amino acid sequence shown in 29.
  • the H-FR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • H-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 .
  • X 1 VQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:29), wherein X 1 can be A or E.
  • the H-FR1 of the antigen binding protein may comprise SEQ ID NO:9 or the amino acid sequence shown in SEQ ID NO:9.
  • the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO:32 amino acid sequence.
  • the H-FR2 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • H-FR2 of the antigen-binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 , X 3 , X 4 .
  • MEWYRQAPGK[X 1 ][X 2 ]ELVAH (SEQ ID NO:32), wherein X 1 can be G or Q, and X 2 can be L or R.
  • the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10.
  • the isolated antigen binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO:31 amino acid sequence.
  • the H-FR3 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • H-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 , X 3 , X 4 .
  • the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:18 or SEQ ID NO:27.
  • the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 may comprise SEQ ID NO : the amino acid sequence shown in 33.
  • the H-FR4 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • H-FR4 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 .
  • WGQGT[X 1 ]VTVSS (SEQ ID NO: 33), wherein X 1 can be M or Q.
  • the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  • the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 29, wherein The H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:32, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:33 amino acid sequence.
  • the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise SEQ ID NO:4 or SEQ ID NO:9.
  • the amino acid sequence shown the H-FR2 can include the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 5
  • the H-FR3 can include SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  • the isolated antigen binding protein may comprise an antigen binding protein selected from any group of H-FR1, H-FR2, H-FR3 and H-FR4: for example,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:7;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18
  • the amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12; or,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:27
  • the amino acid sequence shown and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:12.
  • the heavy chain variable region of the isolated antigen binding protein can comprise at least one CDR in the antibody heavy chain variable region VH and at least one H-FR in the framework region.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, and the C-terminus of the H-FR1 is connected to the The N-terminal of the HCDR1 is directly or indirectly connected, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminal of the H-FR4 directly or indirectly connected to the C-terminus of the HCDR3.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, and the HCDR1 may comprise [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]An amino acid sequence shown in NYA, wherein X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, X 5 is S, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:29
  • the amino acid sequence of the H-FR2 can comprise the amino acid sequence shown in SEQ ID NO:32, the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:31 The amino acid sequence shown in NO:
  • said isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, wherein said HCDR1 may comprise SEQ ID NO:3 , SEQ ID NO:20 and/or the amino acid sequence shown in SEQ ID NO:22, the HCDR2 can include the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 can include the amino acid sequence shown in SEQ ID NO:1 Sequence, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4 and/or SEQ ID NO:9, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10 and/or SEQ ID NO:5 The amino acid sequence of the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 18 and/or SEQ ID NO: 27, and the The H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7
  • the isolated antigen binding protein may comprise an antigen binding protein selected from any group of HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4: for example,
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 amino acid sequence
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14 amino acid sequence
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 amino acid sequence
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14 amino acid sequence
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 amino acid sequence
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 amino acid sequence
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12; or,
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11
  • the amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18
  • the amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:22
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18
  • the amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18
  • the amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1,
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:27
  • the amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the isolated antigen-binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the antigen-binding protein may comprise a VH having amino acid substitutions at one or more amino acids selected from the group (eg, conservative amino acid substitutions, etc.) compared to the sequence shown in SEQ ID NO: 34 : X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 .
  • X 1 can be A or E
  • X 2 can be G or R
  • X 3 can be F or S
  • X 4 can be I or T
  • X 5 can be F
  • X 6 can be S
  • X 7 can be G or Q
  • X 8 can be L or R
  • X 9 can be A or V
  • X 10 can be A, N or S
  • X 11 can be L or V
  • X 12 can be K or R
  • X 13 can be A or P
  • the heavy chain variable region VH of the antigen binding protein may comprise Ab0 (amino acid sequence shown in SEQ ID NO: 8), Ab1 (amino acid sequence shown in SEQ ID NO: 13), Ab2 (amino acid sequence shown in SEQ ID NO: 13), Ab2 (amino acid sequence shown in SEQ ID NO: ID NO:15), Ab3 (amino acid sequence as shown in SEQ ID NO:16), Ab4 (amino acid sequence as shown in SEQ ID NO:17), Ab5 (amino acid sequence as shown in SEQ ID NO:19), Ab6 (amino acid sequence shown in SEQ ID NO: 21), Ab7 (amino acid sequence shown in SEQ ID NO: 23), Ab8 (amino acid sequence shown in SEQ ID NO: 24), Ab9 (amino acid sequence shown in SEQ ID NO 25), the amino acid sequence shown in any one of Ab10 (amino acid sequence as shown in SEQ ID NO: 26) or Ab11 (amino acid sequence as shown in SEQ ID NO: 28
  • the isolated antigen binding protein can include an antigen binding fragment having the same HCDRs (eg, having the same HCDR1-3) as it.
  • the isolated antigen-binding protein may comprise a heavy chain constant region
  • the heavy chain constant region may comprise a constant region derived from IgG, IgM, IgA, IgD and/or IgE.
  • the heavy chain constant region may comprise an IgG-derived constant region.
  • the isolated antigen-binding protein wherein the heavy chain constant region may comprise a constant region derived from human IgG.
  • the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and/or SEQ ID NO:38.
  • the isolated antigen-binding protein can also compete with a reference antibody for binding to the human Siglec15 protein, and the reference antibody can comprise a heavy chain variable region VH, and the VH can comprise HCDR1, HCDR2 and/or Or at least one, two or three of HCDR3.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the sequence of the HCDR3 of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the sequence of the HCDR2 of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence represented by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, and X 5 is S.
  • the sequence of HCDR1 of the reference antibody can be defined according to the IMGT coding system.
  • the HCDR1 of the reference antibody has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 ,X 3 ,X 4 ,X 5 .
  • X 1 can be G or R
  • X 2 can be F or S
  • X 3 can be It is I or T
  • X 4 can be F
  • X 5 can be S.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 22.
  • the HCDR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown as [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein X 1 is G or R, and X 2 is F or S, X3 is I or T, X4 is F, and X5 is S; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1 amino acid sequence.
  • the reference antibody may comprise antibodies Ab0 to Ab11 or an antigen binding protein having the same HCDRs therewith (eg, having the same HCDR1-3 as them).
  • the reference antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the reference antibody may comprise a VH having amino acid substitutions at one or more amino acids selected from the group (eg, conservative amino acid substitutions, etc.) compared to the sequence shown in SEQ ID NO: 34 : X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 .
  • X 1 can be A or E
  • X 2 can be G or R
  • X 3 can be F or S
  • X 4 can be I or T
  • X 5 can be F
  • X 6 can be S
  • X 7 can be G or Q
  • X 8 can be L or R
  • X 9 can be A or V
  • X 10 can be A, N or S
  • X 11 can be L or V
  • X 12 can be K or R
  • X 13 can be A or P
  • the heavy chain variable region of the reference antibody may comprise Ab0 (amino acid sequence shown in SEQ ID NO: 8), Ab1 (amino acid sequence shown in SEQ ID NO: 13), Ab2 (amino acid sequence As shown in SEQ ID NO:15), Ab3 (amino acid sequence as shown in SEQ ID NO:16), Ab4 (amino acid sequence as shown in SEQ ID NO:17), Ab5 (amino acid sequence as shown in SEQ ID NO:19 ), Ab6 (amino acid sequence as shown in SEQ ID NO:21), Ab7 (amino acid sequence as shown in SEQ ID NO:23), Ab8 (amino acid sequence as shown in SEQ ID NO:24), Ab9 (amino acid sequence as shown in SEQ ID NO:24)
  • the reference antibody may comprise a heavy chain constant region, which may comprise an IgG-derived constant region and/or an IgY-derived constant region.
  • the heavy chain constant region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
  • the application provides one or more polypeptides, which may comprise the isolated antigen binding proteins of the application and fragments thereof.
  • one or more immunoconjugates which may comprise the isolated antigen-binding protein or the polypeptide described herein.
  • the immunoconjugate can include other polypeptides, therapeutic agents, probes and/or other antibodies fused or conjugated to the antigen binding protein.
  • the immunoconjugate can include a pharmaceutically acceptable label, detection agent, and/or therapeutic.
  • the application provides one or more isolated nucleic acid molecules that encode the isolated antigen binding proteins described herein or the polypeptides described herein.
  • each of the one or more nucleic acid molecules may encode the entirety of the antigen binding protein, or may encode a portion of the antigen binding protein.
  • each of the one or more nucleic acid molecules may constitute a permutation of one or more nucleic acid sequences.
  • each of the one or more nucleic acid molecules may encode one or more permutations and combinations of antigen binding proteins or fragments thereof described herein.
  • each nucleic acid molecule in one or more nucleic acid molecules can encode any complete HCDR1-HCDR3, FR1-FR4, IgG1-3 heavy chain constant region or one or more permutations and combinations of their fragments.
  • the combination of products encoded by the nucleic acid molecules may be a functional isolated antigen binding protein described herein (e.g., the isolated Antigen binding proteins can specifically bind Siglec15).
  • each nucleic acid of the nucleic acid molecules described herein can be isolated.
  • the nucleic acid molecule can be produced or synthesized by (i) amplification in vitro, e.g., by polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii) purified, e.g., by Enzymatic digestion, gel electrophoresis fractionation and/or gel purification kit recovery, or (iv) synthetic, such as by artificial chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
  • the application provides one or more isolated vectors, which may include one or more nucleic acid molecules and/or other genes described herein.
  • the one or more isolated vectors can include phage vectors, viral vectors and/or bacterial vectors.
  • the one or more isolated vectors may be covalently closed circular double-stranded.
  • the one or more isolated vectors can enter a host cell and replicate autonomously.
  • said other genes may include appropriate marker genes and/or control elements allowing their presence in the host cell and/or selection under appropriate conditions.
  • control elements may include: an origin of replication, a ribosome binding site, a promoter, an enhancer, a silencer, a multiple cloning site, and/or a termination signal.
  • the promoters may include: promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, and/or CMV promoters.
  • the marker gene may include: an antibiotic resistance gene, a luciferase marker gene and/or a ⁇ -galactosidase gene.
  • the application provides one or more cells.
  • the cells may comprise the isolated antigen binding proteins and/or fragments thereof, the polypeptides, the immunoconjugates, the one or more nucleic acid molecules and/or the described nucleic acid molecules described herein.
  • the vectors described above may comprise one, one, a plurality and/or more of the nucleic acid molecules or vectors described herein.
  • a vector described herein can be introduced into the host cell.
  • the cells may include: prokaryotic cells (e.g., E.
  • the Methods known in the art introduce the carrier described in the application into the host cell, and the method may include: heat shock transformation, transient transfection and/or stable transfection.
  • the transfection may Including: electroporation, liposome transfection and/or calcium phosphate transfection, etc.
  • the cells may include introducing the vectors described in the present application into the cells by methods known in the art.
  • Express for example, transiently express and/or stably express
  • the antigen binding protein or fragment thereof and/or other protein described in the application in said host cell can transiently express the antigen-binding protein described in the present application.
  • the cells for example, CHOK1 cells
  • the cells can stably express human Siglec protein.
  • the cells can include CHOK1 cells.
  • Described cell can comprise the escherichia coli cell of competence.
  • described cell can comprise mammalian cell.
  • described cell can comprise immune cell.
  • described immune cell can comprise: Peripheral blood mononuclear cell (PBMC cell ), T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes and/or leukocytes.
  • PBMC cell Peripheral blood mononuclear cell
  • T cells T cells
  • B cells natural killer cells
  • macrophages NKT cells
  • monocytes monocytes
  • dendritic cells granulocytes
  • lymphocytes and/or leukocytes.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or pharmaceutically acceptable adjuvants and / or excipients.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional media or reagents are contemplated for use in the pharmaceutical compositions of the present application.
  • the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, and may also be referred to as auxiliary materials.
  • the excipients may include binders, fillers, disintegrants, lubricants in tablets.
  • the excipients may include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills.
  • the excipient may comprise the base part of a semi-solid formulation ointment, cream.
  • the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.
  • the present application provides a pharmaceutical combination comprising the isolated antigen-binding protein described in the present application and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor may include substances that inhibit the interaction of PD-1/PD-L1.
  • the immune checkpoint inhibitor can be selected from the group consisting of PD-1/PD-L1 blockers, PD-1 antagonists, PD-L1 antagonists, PD-1 inhibitors and PD-L1 inhibitors.
  • the PD-1/PD-L1 blocking agent can be selected from the group consisting of BMS202 (PD-1/PD-L1 inhibitor 2), BMS-1 (PD-1/PD-L1 inhibitor 1), PD-1/PD-L1 Inhibitor 3, BMS-1166 and BMS-1001.
  • the PD-1 inhibitor can include an anti-PD-1 antibody.
  • the PD-L1 inhibitor can include an anti-PD-L1 antibody.
  • the anti-PD-1 antibody can be selected from the group consisting of Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimumab) ), Sintilimab (Sintilimab) and Tislelizumab (Tislelizumab).
  • the anti-PD-L1 antibody may be selected from the group consisting of Durvalumab (Dervalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-1 antibody may comprise HCDR3 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise HCDR2 of an antibody selected from the following group: Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise HCDR1 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise LCDR3 of an antibody selected from the following group: Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise LCDR2 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise LCDR1 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise the VH of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-1 antibody may comprise the VL of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Carrelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
  • the anti-PD-L1 antibody may comprise HCDR3 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise HCDR2 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise HCDR1 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise LCDR3 of an antibody selected from the group consisting of Durvalumab (Dirvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise LCDR2 of an antibody selected from the group consisting of Durvalumab (Dirvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise LCDR1 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise the VH of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-L1 antibody may comprise the VL of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
  • the anti-PD-1 antibody may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:39.
  • the anti-PD-1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:40.
  • the anti-PD-1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41.
  • the anti-PD-1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 39; the HCDR2 comprises SEQ ID NO: 39 The amino acid sequence shown in ID NO:40; And described HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41.
  • the anti-PD-1 antibody can comprise pembrolizumab or an antibody that has the same HCDR3 as it (eg, has the same HCDR1-3 as it).
  • the anti-PD-1 antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:42.
  • the anti-PD-1 antibody may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.
  • the anti-PD-1 antibody may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:44.
  • the anti-PD-1 antibody may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:45.
  • the anti-PD-1 antibody may comprise a light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 43; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:44; and said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:45.
  • the anti-PD-1 antibody can comprise pembrolizumab or an antibody that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
  • the anti-PD-1 antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the anti-PD-1 antibody may comprise a heavy chain and a light chain, the heavy chain may comprise HCDR1-3 and H-FR1-4, and the light chain may comprise LCDR1-3 and L-FR1 -4.
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:45; the LCDR2 can include the amino acid sequence shown in SEQ ID NO:44; the LCDR3 can include the amino acid sequence shown in SEQ ID NO:43.
  • the anti-PD-1 antibody can comprise pembrolizumab or an antigen binding protein that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as it.
  • the heavy chain variable region of the anti-PD-1 antibody may comprise the amino acid sequence shown in SEQ ID NO:42.
  • the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the anti-PD-1 antibody may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same light chain variable region as it.
  • the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same heavy chain as it.
  • the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same light chain as it.
  • the anti-PD-L1 antibody may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:47.
  • the anti-PD-L1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:48.
  • the anti-PD-L1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49.
  • the anti-PD-L1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 47; the HCDR2 comprises SEQ ID NO: 47 The amino acid sequence shown in ID NO:48; And described HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49.
  • the anti-PD-L1 antibody can comprise atezolizumab or an antibody that has the same HCDR3 as it (eg, has the same HCDR1-3 as it).
  • the anti-PD-L1 antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:50.
  • the anti-PD-L1 antibody may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:51.
  • the anti-PD-L1 antibody may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:52.
  • the anti-PD-L1 antibody may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:53.
  • the anti-PD-L1 antibody may comprise a light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 51; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:52; and said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:53.
  • the anti-PD-L1 antibody can comprise atezolizumab or an antibody that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
  • the anti-PD-L1 antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the anti-PD-L1 antibody may comprise a heavy chain and a light chain, the heavy chain may comprise HCDR1-3 and H-FR1-4, and the light chain may comprise LCDR1-3 and L-FR1 -4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:49; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:48; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:47;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:53; the LCDR2 can include the amino acid sequence shown in SEQ ID NO:52; the LCDR3 can include the amino acid sequence shown in SEQ ID NO:51.
  • the anti-PD-L1 antibody can comprise atezolizumab or an antigen binding protein that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as atezolizumab.
  • the heavy chain variable region of the anti-PD-L1 antibody may comprise the amino acid sequence shown in SEQ ID NO:50.
  • the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the anti-PD-L1 antibody may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same light chain variable region as it.
  • the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same heavy chain as it.
  • the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same light chain as it.
  • the drug combination described in this application can effectively kill tumors.
  • each active ingredient in the pharmaceutical combination can be administered in combination.
  • an isolated antigen binding protein described herein can be administered in combination with the immune checkpoint inhibitor.
  • the combined administration can be administered simultaneously or sequentially.
  • the combined administration may be that each active ingredient is independently administered once, or each active ingredient is independently administered multiple times.
  • the present application provides a method for detecting or measuring Siglec15.
  • the method may comprise the use of an isolated antigen binding protein described herein.
  • the methods may include: in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the method may include a method for detecting the presence and/or amount of Siglec protein for non-diagnostic or non-therapeutic purposes, which may include the steps of:
  • the present application provides a detection kit for detecting Siglec protein.
  • the detection kit can include an isolated antigen binding protein described herein.
  • the detection kit may include instructions for use, one or more containers, chemical reagents and/or biomacromolecules.
  • the present application provides a use of the isolated antigen-binding protein in preparing a kit, and the kit can be used in a method for detecting the presence and/or content of Siglec protein.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the present application provides a method of modulating an immune response.
  • the method may comprise administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell , the pharmaceutical composition, the pharmaceutical combination, a pharmaceutically acceptable therapeutic agent and/or a pharmaceutically acceptable adjuvant and/or excipient.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the diseases or conditions may include: tumors.
  • the tumor can include a tumor associated with expression of Siglec15.
  • the term "tumor associated with the expression of Siglec15" generally refers to the formation of tumors in which the expression of Siglec15 protein is up-regulated in the tumor microenvironment to escape from the immune system.
  • the tumors associated with the expression of Siglec15 may be Siglec15 positive tumors.
  • the expression of Siglec15 protein on the surface of immune system cells in the tumor microenvironment is about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher.
  • the tumor can comprise a solid tumor.
  • the present application provides a method for preventing and/or treating a disease or disorder, the method may comprise administering the isolated antigen-binding protein, the immunoconjugate described in the present application to a subject in need compound, the isolated nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination.
  • the diseases or conditions may include: tumors.
  • the tumor can include a tumor associated with expression of Siglec15.
  • the tumor can include a tumor associated with expression of Siglec15.
  • the term "tumor associated with the expression of Siglec15" generally refers to the formation of tumors in which the expression of Siglec15 protein is up-regulated in the tumor microenvironment to escape from the immune system.
  • the tumors associated with the expression of Siglec15 may be Siglec15 positive tumors.
  • the expression of Siglec15 protein on the surface of immune system cells in the tumor microenvironment is about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher.
  • the tumor can comprise a solid tumor.
  • the application provides the isolated antigen binding protein, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination , for the prevention and/or treatment of a disease or condition.
  • the diseases or conditions may include: tumors.
  • the tumor can include a tumor associated with expression of Siglec15.
  • the tumor can include a tumor associated with expression of Siglec15.
  • the term "tumor associated with the expression of Siglec15" generally refers to the formation of tumors in which the expression of Siglec15 protein is up-regulated in the tumor microenvironment to escape from the immune system.
  • the tumors associated with the expression of Siglec15 may be Siglec15 positive tumors.
  • the expression of Siglec15 protein on the surface of immune system cells in the tumor microenvironment is about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher.
  • the tumor can comprise a solid tumor.
  • the subjects in need may include humans or other animals.
  • the other animals may include: monkeys, chickens, geese, cats, dogs, mice and/or rats.
  • other animals may also include any animal species other than humans, eg, livestock animals, poultry animals, rodents, and/or primates.
  • the human can be Caucasian, African, Asian, Semitic and/or other ethnicity.
  • the human can be elderly, adult, adolescent, child or infant.
  • Embodiment 1 immune alpaca
  • a healthy female adult alpaca (Alpaca) was immunized with recombinant human Siglec15 protein (SG5-H5253, ACRO).
  • 500 ⁇ g recombinant human Siglec15 protein was emulsified with an equal volume of Freund’s complete adjuvant and injected on the left and right sides near the cervical lymph nodes;
  • 500 ⁇ g recombinant human Siglec15 protein was emulsified with an equal volume of Freund’s complete or incomplete adjuvant and then injected in The left and right sides of the neck lymph nodes were injected, and several booster immunizations were carried out. Blood was collected one week after the last immunization to test the antiserum titer.
  • the peripheral blood of the alpaca immunized in Example 1 was collected, and the lymphocytes in the peripheral blood of the alpaca immunized in Example 1 were obtained by using a lymphocyte separation medium; the total RNA of the lymphocytes was extracted by using TRIzol TM Reagent; The reverse transcription kit (PrimeScript TM II 1st Strand cDNA Synthesis Kit) was reverse transcribed to obtain cDNA, and the VHH gene of the alpaca antibody was amplified by nested PCR.
  • the reverse transcription kit (PrimeScript TM II 1st Strand cDNA Synthesis Kit) was reverse transcribed to obtain cDNA, and the VHH gene of the alpaca antibody was amplified by nested PCR.
  • the VHH gene fragment was recovered with a gel purification kit, digested with restriction endonuclease Bgl1, and then cloned into the phagemid vector pADL-10b, and the constructed clone product was transformed into E.coli TG1 electroporation competent Cells were used to construct a VHH gene library; the library capacity was determined to be 2.2 ⁇ 10 9 pfu by the plate gradient dilution method, and the colony PCR results showed that the library insertion rate was 97.9%. Take live cells with 10-100 times the library capacity from the above-mentioned gene library for inoculation and culture, and use M13K07 phage to rescue after culturing to the logarithmic phase. After the rescue culture, collect the phage by centrifugation, and use PEG-NaCl to purify the phage to obtain phage display. Libraries can be used directly for subsequent screening.
  • the screened biologically active antibodies were humanized.
  • the humanization of camel monoclonal antibodies is carried out according to the methods published in many documents in this field.
  • Human antibody constant domains can be used to replace the parental (camel antibody) constant domains, and human germline antibody sequences can be selected according to the homology between camel antibodies and human antibodies for CDR grafting.
  • the constant region of the camel antibody can be replaced by a human constant region through back-mutation of the amino acid residues of the VH of the camel antibody to obtain the final humanized binding protein.
  • the antigen-binding proteins after humanization in this example are shown in Table 1.
  • Table 1 The variable region sequence information of the humanized antigen-binding protein
  • a plasmid was constructed according to the sequence of the antigen-binding protein obtained in Example 4, which was transiently expressed in Expi293 cells (Thermo Fisher, catalog number: A14527CN), and the antibody expression plasmid was extracted using a large-scale extraction kit.
  • Solution 1 Dilute 15 ⁇ g plasmid with 1ml culture medium and mix well.
  • Solution 2 Dilute 60 ⁇ l transfection reagent with 1ml culture medium and mix well. Add solution 2 to solution 1, mix well, and incubate at 37°C for 15 minutes, add the mixed transfection solution dropwise to the cell solution, add while shaking, put it on a shaker for culture, express for one week, collect the supernatant, 8000 rpm /min centrifuge for 5 minutes.
  • Dialysis suck the high-concentration protein into a dialysis bag and put it in a beaker of 1 ⁇ PBS for dialysis.
  • the purity test using high performance liquid chromatography LC-20AT and gel chromatographic column is qualified, and the endotoxin test is qualified.
  • the binding ability of the Siglec15 antigen-binding protein of Ab0 (amino acid sequence shown in SEQ ID NO: 8) obtained in Example 5 to human Siglec15 expressed on the surface of CHOK1 cells was determined based on flow cytometry.
  • the binding ability of different Siglec15 antigen-binding proteins was determined by comparing the binding curves of human Siglec15 expressed on the surface of CHOK1 cells.
  • the cells were resuspended in PBS with 2% FBS, and the median fluorescence value (MFI) of the PE channel was measured by flow cytometry.
  • MFI median fluorescence value
  • Example 5 of the present application has binding activity to human Siglec15 overexpressed on CHOK1 cells, and is superior to positive antibodies.
  • the results of the binding curve of Siglec15 antigen-binding protein and human Siglec15 expressed on the surface of CHOK1 cells based on flow cytometry are shown in FIG. 1 .
  • Table 2 shows the EC50 value and the highest MFI value of Siglec15 antigen-binding protein binding to human Siglec15 expressed on the surface of CHOK1 cells based on flow cytometry.
  • PBMC peripheral mononuclear cells
  • FACS buffer PBS+2% FBS
  • APC anti-human CD8 BioLegend, 344722
  • PE-Cy7 anti-human CD4 BioLegend, 357410 antibodies were diluted with FACS buffer, and the diluted samples were added to a 96-well round bottom plate and incubated at room temperature for 30 min. Centrifuge at 400g for 5min and discard the supernatant.
  • the above tests show that the Siglec15 antigen-binding protein of the present application can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the blood of subjects.
  • the experimental results are shown in Figure 2A-C, Siglec15 antigen-binding protein can promote the proliferation of CD8 and CD4 T cells and the expression of IFN- ⁇ in the culture supernatant, and its activity is equivalent to or better than that of the positive antibody.
  • the experimental results are shown in Table 3.
  • the Siglec15 antigen binding protein can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the subject's blood.
  • Table 3 The ability of Siglec15 binding to block Siglec15 to inhibit the proliferation of PBMC in the blood of subjects
  • the binding ability of Siglec15-binding protein to human Siglec15 expressed on the surface of CHOK1 cells was determined based on a flow cytometry assay.
  • the binding ability of different humanized Siglec15 binding proteins of the present application was determined by comparing the binding curves of human Siglec15 expressed on the surface of CHOK1 cells.
  • the cells were resuspended in PBS with 2% FBS, and the median fluorescence value (MFI) of the PE channel was measured by flow cytometry.
  • MFI median fluorescence value
  • the above tests show that the humanized Siglec15-binding protein of the present application has binding activity to human Siglec15 overexpressed on CHOK1 cells, and is superior to positive antibodies.
  • the experimental results are shown in Figure 3A-B, the binding curve of humanized Siglec15 binding protein and human Siglec15 expressed on the surface of CHOK1 cells was determined based on flow cytometry.
  • the experimental results are shown in Table 4. The EC50 value and the highest MFI value of the humanized Siglec15-binding protein binding to human Siglec15 expressed on the surface of CHOK1 cells were determined based on flow cytometry.
  • Table 4 EC 50 value and highest MFI value of humanized Siglec15-binding protein binding to Siglec15 expressed on the surface of CHOK1 cells
  • PBMC peripheral mononuclear cells
  • FACS buffer PBS+2% FBS
  • APC anti-human CD8 BioLegend, 344722
  • PE-Cy7 anti-human CD4 BioLegend, 357410 antibodies were diluted with FACS buffer, and the diluted samples were added to a 96-well round bottom plate and incubated at room temperature for 30 min. Centrifuge at 400g for 5min and discard the supernatant.
  • the above experiments show that the humanized Siglec15 antigen-binding protein of the present application can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the subject's blood.
  • the experimental results are shown in Figure 4A-C, the humanized Siglec15 antigen-binding protein can promote the proliferation of CD8 and CD4 T cells and the quantitative expression of IFN- ⁇ in the culture supernatant, and its activity is better than that of the positive antibody.
  • the experimental results are shown in Table 5.
  • the Siglec15 antigen-binding protein can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the subject's blood.
  • Siglec15 antigen-binding protein blocks the ability of Siglec15 to inhibit the proliferation of PBMC in the blood of subjects
  • Siglec15 antigen-binding proteins The binding affinity of different Siglec15 antigen-binding proteins to antigen (Siglec15 protein, human, recombinant (ECD, His Tag), source: Acro, catalog number: SG5-C5253) protein was detected by octet RED 384.
  • Reagent preparation 100mlPBS (gibco, catalog number 10010-023) was added to 20ul Tween 20 (SIGMA, catalog number P1379-500ML), and 220 ⁇ l per well was added to the well plate of Bio-One 96-well black flat-bottom (Greiner, catalog number 655209). in the corresponding position.
  • Regeneration Solution Add 220 ⁇ l per well of Glycine solution (10mM, PH 1.7) to the corresponding position of the Bio-One 96-well black flat-bottom well plate.
  • Sample preparation Dilute the positive control antibody, negative antibody to be tested and Siglec15 antigen-binding protein to be tested with PBST (0.02% Tween20) to 5 ⁇ g/ml, set up experimental group and reference group for each Siglec15 antigen-binding protein, add 220 ⁇ l per well to Bio- In the corresponding position of the One 96-well black flat-bottom orifice plate.
  • PBST 0.02% Tween20
  • Antigen preparation Dilute human Siglec15-His to 100nM with PBST (0.02% Tween20), and add 220 ⁇ l per well to the corresponding position of the Bio-One 96-well black flat-bottom well plate.
  • This test detects the interaction between Siglec15 antigen binding protein and human Siglec15-his (Acro, Cat. No. 5G5-H52H3), cynomolgus Siglec15-hFc (Acro, Cat. protein binding.
  • the above tests show that the humanized Siglec15 antigen-binding protein of the present application has the ability to bind human, monkey and mouse Siglec15 antigens.
  • the experimental results are shown in Figure 5A-F, the humanized Siglec15 antigen binding protein has binding curves to human, monkey and mouse Siglec15 antigens.
  • the experimental results are shown in Table 7, the binding ability of humanized Siglec15 antigen binding protein to human, monkey and mouse Siglec15 antigen.
  • Table 7 The binding ability of humanized Siglec15 antigen-binding protein to human, monkey and mouse Siglec15 antigen
  • MC38/hSiglec15 cells were used to inoculate C57BL/6N mice to determine the anti-tumor effect of the Siglec15 antigen-binding protein of the present invention.
  • C57BL/6N mice female C57BL/6N mice (6 weeks) (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.). Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
  • mice MC38 cells mouse MC38 cells (Heyuan Biotechnology (Shanghai) Co., Ltd.) were routinely subcultured according to the instructions; SPH genetically modified MC38 cells to overexpress human Siglec15, and the cells were named MC38/hSiglec15 cells for subsequent in vivo experiments. The cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of wild-type C57BL/6N mice to establish the MC38/hSiglec15 tumor-bearing mouse model.
  • mice with a tumor volume in the range of 100 mm 3 to 190 mm 3 were selected and divided into groups according to the average tumor volume (6 mice in each group).
  • negative control antibody human IgG
  • positive control antibody SPH-Sg-PC-1, Taizhou Baiying Biotechnology Co., Ltd., catalog number B218501
  • the tumor volume and weight changes of the mice in each group were monitored during the administration period, and the monitoring frequency was 2 times/week, and the monitoring was continued for 3 weeks.
  • TGI tumor volume inhibition rate
  • FIG. 6A The experimental results of the anti-tumor efficacy of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 in wild-type C57BL/6N mice are shown in Figure 6A. After administration of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4, the experimental results of body weight changes in wild-type C57BL/6N mice are shown in Figure 6B. On the 24th day after inoculation, the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application significantly inhibited the growth of tumors at a dose of 10 mg/kg, and the tumor volume inhibition rates were 97%, 89%, 100%, and 95%. and 104%, the experimental results are shown in Fig. 6C.
  • the ability of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 to inhibit tumor growth in vivo on day 24 is shown in Table 9.
  • the above experiments show that the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application significantly inhibit the growth of tumors, and have no significant effect on the body weight of wild-type C57BL/6N mice.
  • MC38/hSiglec15 cells were used to inoculate human Siglec15 transgenic mice to determine the anti-tumor effect of the Siglec15 antigen-binding protein of the present invention.
  • Human Siglec15 transgenic mice female human Siglec15 transgenic mice (6 weeks) of C57BL/6 background (Shanghai Southern Model Biotechnology Co., Ltd.). Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
  • Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and conventionally subcultured according to the instructions; MC38 cells were genetically modified to overexpress human Siglec15, and the cells were named MC38/human Siglec15 cells for subsequent in vivo experiments. The cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of human Siglec15 transgenic mice to establish the MC38/human Siglec15 tumor-bearing mouse model.
  • TGI tumor volume inhibition rate
  • FIG. 7A The results of the anti-tumor efficacy test of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 in the human Siglec15 transgenic mice in the first experiment are shown in Figure 7A. After administration of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 in the first experiment, the results of the body weight change experiment of the human Siglec15 transgenic mice are shown in Fig. 7B. The results of the anti-tumor efficacy test of the antigen-binding proteins Ab0, Ab5 and Ab10 in the human Siglec15 transgenic mice in the second experiment are shown in FIG. 8A . After administration of the antigen-binding proteins Ab0, Ab5 and Ab10 in the second experiment, the results of the body weight change experiment of the human Siglec15 transgenic mice are shown in FIG. 8B .
  • the experimental results are shown in Table 11 and Table 12.
  • the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application significantly inhibited tumor growth at a dose of 10 mg/kg.
  • the tumor volume inhibition rates were 87%, 60%, 51%, 75% and 83%, respectively.
  • the antigen-binding proteins Ab0, Ab5 and Ab10 of the present application significantly inhibited the growth of tumors at a dose of 10 mg/kg, and the tumor volume inhibition rates were 55%, 70% and 32% respectively .
  • Antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application have the ability to inhibit tumor growth in vivo on the 25th day in the first test
  • Antigen-binding proteins Ab0, Ab5 and Ab10 of the present application have the ability to inhibit tumor growth in vivo on the 23rd day in the second test
  • PBMC peripheral mononuclear cells
  • the humanized Siglec15 antigen-binding protein can inhibit the formation of osteoclasts, and the activity is comparable to that of the positive antibody.
  • MC38/hSiglec15 cells were used to inoculate human PD-1/PD-L1 transgenic mice to determine the anti-tumor effect of Siglec15 antigen-binding protein of the present invention combined with PD-1 antibody.
  • Human PD-1/PD-L1 transgenic mice Female human PD-1/PD-L1 transgenic mice (6 weeks old) with C57BL/6 background were purchased from Shangbai Ocyto Jiangsu Gene Biotechnology Co., Ltd. Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
  • Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and conventionally subcultured according to the instructions; MC38 cells were genetically modified to overexpress human Siglec15, and the cells were named MC38/human Siglec15 cells for subsequent in vivo experiments. Cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of human PD-1/PD-L1 transgenic mice to establish MC38/human Siglec15 loading. Tumor mouse model.
  • mice with a tumor volume in the range of 100 mm 3 to 235 mm 3 were selected and grouped according to the average tumor volume (6 mice in each group).
  • PD-1 antibody pembrolizumab, source: Taizhou Baiying Biotechnology Co., Ltd., product number: B2014-CHO
  • negative control antibody human IgG
  • positive control antibody SPH-Sg-PC-1, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number HSP067-41
  • the changes in tumor volume and body weight of mice in each group were monitored at a frequency of 2 times/week for 3 consecutive weeks.
  • TGI (%) [1-(Ti-T0)/(Vi-V0) ] ⁇ 100;
  • Ti average tumor volume of the administration group
  • T0 average tumor volume of the administration group on D0 day
  • Vi average tumor volume of the same-type control group
  • V0 average tumor volume of the same-type control group on D0 day .
  • Body weight was measured using an electronic balance.
  • the tumor volume inhibition rates of the antigen-binding proteins Ab5 and Ab10 of the present application at a dose of 10 mg/kg were -1% and 11%, respectively, and the PD-1 antibody at a dose of 1 mg/kg
  • the tumor volume inhibition rate was 9%; the tumor volume inhibition rate was 67% under the combined dose of Ab5 and PD-1 antibody; the tumor volume inhibition rate was 63% under the combined dose of Ab10 and PD-1 antibody;
  • the combination of protein Ab5 and Ab10 with PD-1 antibody can significantly inhibit the growth of tumor.
  • MC38/hSiglec15 cells were used to inoculate human PD-1/PD-L1 transgenic mice to determine the anti-tumor effect of Siglec15 antigen-binding protein of the present invention combined with PD-L1 antibody.
  • Human PD-1/PD-L1 transgenic mice Female human PD-1/PD-L1 transgenic mice (6 weeks old) with C57BL/6 background were purchased from Shangbai Osset Jiangsu Gene Biotechnology Co., Ltd. Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
  • Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and conventionally subcultured according to the instructions; MC38 cells were genetically modified to overexpress human Siglec15, and the cells were named MC38/human Siglec15 cells for subsequent in vivo experiments. Cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of human PD-1/PD-L1 transgenic mice to establish MC38/human Siglec15 loading. Tumor mouse model.
  • mice with a tumor volume in the range of 120 mm 3 to 160 mm 3 were selected and divided into groups according to the tumor volume (6 mice in each group).
  • negative control antibody human IgG
  • PD-L1 antibody atezolizumab, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number: HSP099-20
  • the control antibody SPH-Sg-PC-1, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number HSP067-41
  • the antigen-binding protein of the application and the changes in tumor volume and body weight of mice in each group were monitored during the administration, and the monitoring frequency was uniform.
  • TGI (%) [1-(Ti-T0)/(Vi-V0) ] ⁇ 100;
  • Ti average tumor volume of the administration group
  • T0 average tumor volume of the administration group on D0 day
  • Vi average tumor volume of the same-type control group
  • V0 average tumor volume of the same-type control group on D0 day .
  • Body weight was measured using an electronic balance.
  • the tumor volume inhibition rates of the antigen-binding proteins Ab5 and Ab10 of the present application at a dose of 10 mg/kg were 91% and 63%, respectively, and the PD-L1 antibody at a dose of 3 mg/kg
  • the tumor volume inhibition rate was 58%; the tumor volume inhibition rate was 101% under the combined dose of Ab5 and PD-L1 antibody; the tumor volume inhibition rate was 98% under the combined dose of Ab10 and PD-L1 antibody; the antigen binding protein of the application Both Ab5 and Ab10 combined with PD-L1 antibody significantly inhibited tumor growth.

Abstract

Provided are a human Siglec15 antigen-binding protein and a use thereof. The human Siglec15 antigen-binding protein specifically binds to the protein human Siglec15 with a KD value of approximately 5E-09M or lower, and can release Siglec15 from inhibiting the proliferation of PBMCs in blood, thereby inhibiting the growth and/or proliferation of tumor cells. Also provided are an immunoconjugate, a polypeptide, a nucleic acid molecule, a carrier and a pharmaceutical composition comprising the Siglec15 antigen-binding protein, a method for detecting Siglec15, and a kit for detecting Siglec15; a method for the Siglec15 antigen-binding protein and a use of the Siglec15 antigen-binding protein are also provided.

Description

靶向Siglec15的抗原结合蛋白及其用途Antigen-binding protein targeting Siglec15 and use thereof 技术领域technical field
本申请涉及生物医药领域，具体的涉及一种靶向Siglec15的抗原结合蛋白及其用途。This application relates to the field of biomedicine, in particular to an antigen-binding protein targeting Siglec15 and its use.
背景技术Background technique
Siglec15是I型跨膜蛋白，包括两个Ig样结构域、一个包含赖氨酸残基的跨膜结构域、一个短的细胞质尾巴。该蛋白最初被鉴定为具有典型唾液酸结合免疫球蛋白样凝集素结构的Siglec基因家族成员。在大多数正常人类组织和各种免疫细胞亚群中，Siglec15的mRNA表达水平极低，但在巨噬细胞中表达较高。Siglec15 is a type I transmembrane protein consisting of two Ig-like domains, a transmembrane domain containing lysine residues, and a short cytoplasmic tail. This protein was originally identified as a member of the Siglec gene family with a typical sialic acid-binding immunoglobulin-like lectin structure. Siglec15 mRNA is expressed at very low levels in most normal human tissues and in various immune cell subsets, but is higher in macrophages.
2019年，陈列平等发现siglec15是一种巨噬细胞相关的T细胞抑制分子，其表达通常局限于髓系细胞，但在人类肿瘤组织中广泛表达。IFN-γ可以抑制Siglec15表达水平，而Siglec15体外抑制T细胞活性。Siglec15敲除小鼠各项生理指标正常，显示了Siglec15靶点的安全性。Siglec15通过抑制IL-10抑制抗原特异性T细胞反应，同时，Siglec15敲除小鼠中CD8+T细胞、NK细胞以及细胞因子分泌增加，肿瘤发生率降低，生存率升高。以上结果提示Siglec15可能是一个非常有潜力的肿瘤治疗靶点。In 2019, Chen Lieping discovered that siglec15 is a macrophage-associated T cell inhibitory molecule, whose expression is usually limited to myeloid cells, but widely expressed in human tumor tissues. IFN-γ can inhibit the expression level of Siglec15, and Siglec15 inhibits T cell activity in vitro. The physiological indicators of Siglec15 knockout mice were normal, showing the safety of the Siglec15 target. Siglec15 inhibits antigen-specific T cell responses by inhibiting IL-10. At the same time, Siglec15 knockout mice increase the secretion of CD8+T cells, NK cells and cytokines, reduce the incidence of tumors, and increase the survival rate. The above results suggest that Siglec15 may be a very potential target for tumor therapy.
目前，靶向Siglec15的抗原结合蛋白还存在选择性不强、亲和力较低的缺陷。因此，有必要开发靶向Siglec15的高亲和力、特异性强的新型抗原结合蛋白。At present, the antigen-binding proteins targeting Siglec15 still have the defects of poor selectivity and low affinity. Therefore, it is necessary to develop novel antigen-binding proteins with high affinity and strong specificity targeting Siglec15.
发明内容Contents of the invention
本申请提供了一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：a)在Biacore检测中，以约5E-09M或以下的K D值与人Siglec15蛋白特异性结合；b)在流式检测中，以约0.2μg/ml或以下的EC50值与表达于CHOK1细胞表面的人Siglec15特异性结合；c)解除Siglec15对血液中PBMC的增殖抑制；d)在ELISA检测中，以约0.2μg/ml或以下的EC50值与人Siglec15特异性结合；e)在ELISA检测中，以约0.1μg/ml或以下的EC50值与猴Siglec15特异性结合；f)在ELISA检测中，以约0.1μg/ml或以下的EC50值与鼠Siglec15特异性结合；以及g)抑制肿瘤细胞的生长和/或增殖。 The application provides an isolated antigen-binding protein, which has one or more of the following properties: a) specifically binds to human Siglec15 protein with a KD value of about 5E-09M or below in a Biacore assay ; b) in flow cytometry, it specifically binds to human Siglec15 expressed on the surface of CHOK1 cells with an EC50 value of about 0.2 μg/ml or below; c) releases Siglec15 from inhibiting the proliferation of PBMCs in blood; d) detects in ELISA Among them, it specifically binds to human Siglec15 with an EC50 value of about 0.2 μg/ml or below; e) In ELISA detection, it specifically binds to monkey Siglec15 with an EC50 value of about 0.1 μg/ml or below; f) In ELISA detection wherein, specifically binds to murine Siglec15 with an EC50 value of about 0.1 μg/ml or less; and g) inhibits the growth and/or proliferation of tumor cells.
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。In certain embodiments, the isolated antigen-binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。In certain embodiments, the isolated antigen-binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR1，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S。 In certain embodiments, the isolated antigen-binding protein comprises HCDR1 comprising the amino acid sequence [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein , X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S.
在某些实施方式中，所述的分离的抗原结合蛋白中所述HCDR1包含SEQ ID NO:3或SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。In some embodiments, the HCDR1 in the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:20 or SEQ ID NO:22.
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。 In certain embodiments, said isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA Amino acid sequence, wherein X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 , and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述的分离的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22, wherein Said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:29所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 29.
在某些实施方式中，所述的分离的抗原结合蛋白中所述H-FR1包含SEQ ID NO:9或SEQ ID NO:9所示的氨基酸序列。In some embodiments, the H-FR1 in the isolated antigen binding protein comprises SEQ ID NO:9 or the amino acid sequence shown in SEQ ID NO:9.
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:32所示的氨基酸序列。In some embodiments, the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:32 amino acid sequence.
在某些实施方式中，所述的分离的抗原结合蛋白中所述H-FR2包含SEQ ID NO:5或SEQ ID NO:10所示的氨基酸序列。In some embodiments, the H-FR2 in the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10.
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:31所示的氨基酸序列。In some embodiments, the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO:31 amino acid sequence.
在某些实施方式中，所述的分离的抗原结合蛋白中所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18或SEQ ID NO:27所示的氨基酸序列。In certain embodiments, said H-FR3 in said isolated antigen binding protein comprises SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: The amino acid sequence shown in 27.
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。In some embodiments, the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 33.
在某些实施方式中，所述的分离的抗原结合蛋白中所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。In some embodiments, the H-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:29所示的氨基酸序列，所述H-FR2包含SEQ ID NO:32所示的氨基酸序列，所述H-FR3包含SEQ ID NO:31所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。In some embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 29, wherein The H-FR2 comprises the amino acid sequence shown in SEQ ID NO:32, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:33 .
在某些实施方式中，所述的分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10或SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18或SEQ ID NO:27所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。In certain embodiments, said isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NO:4 or SEQ ID NO:9 The amino acid sequence shown, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:5, and the H-FR3 comprises SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO: 14. The amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:27, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
在某些实施方式中，所述的分离的抗原结合蛋白包含选自下述任一组H-FR1，H-FR2，H-FR3和H-FR4：1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；2)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；3)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；4)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；5)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；6)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；7)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；8)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:27所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列。In some embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups: 1) the H-FR1 comprises SEQ ID NO The amino acid sequence shown in: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 6 The amino acid sequence shown in ID NO:7; 2) said H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, and said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said H- FR3 comprises the amino acid sequence shown in SEQ ID NO:11, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 3) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, The H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12 sequence; 4) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 The amino acid sequence shown, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 12; 5) the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 9, and the H-FR2 includes the amino acid sequence shown in SEQ ID The amino acid sequence shown in NO:5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO:12; 6) the H- FR1 comprises the amino acid sequence shown in SEQ ID NO:9, said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18, and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 7) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 , the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 18, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12; 8) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 9 The amino acid sequence shown, the H-FR2 includes the amino acid sequence shown in SEQ ID NO:5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:27, and the H-FR4 Comprising the amino acid sequence shown in SEQ ID NO:12.
在某些实施方式中，所述的分离的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ  ID NO:34所示的氨基酸序列。In some embodiments, the isolated antigen-binding protein comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:34.
在某些实施方式中，所述的分离的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:28所示的氨基酸序列。In certain embodiments, said isolated antigen binding protein comprises a heavy chain variable region VH comprising SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16 , SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:28 amino acid sequence.
在某些实施方式中，所述分离的抗原结合蛋白包含重链恒定区，且所述重链恒定区包含源自IgG的恒定区。In certain embodiments, the isolated antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region.
在某些实施方式中，所述分离的抗原结合蛋白中所述重链恒定区包括源自人IgG的恒定区。In certain embodiments, said heavy chain constant region of said isolated antigen binding protein comprises a constant region derived from human IgG.
在某些实施方式中，所述分离的抗原结合蛋白中所述重链恒定区包含SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37或SEQ ID NO:38所示的氨基酸序列。In some embodiments, the heavy chain constant region in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38 .
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或抗原结合片段。In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment.
在某些实施方式中，所述分离的抗原结合蛋白中所述抗原结合片段选自：Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv，VHH和/或dAb。 In certain embodiments, the antigen-binding fragment of the isolated antigen-binding protein is selected from the group consisting of: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
在某些实施方式中，所述的分离的抗原结合蛋白中所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。In certain embodiments, said antibody in said isolated antigen binding protein is selected from the group consisting of monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.
在某些实施方式中，所述的分离的抗原结合蛋白，其为单域抗体。In certain embodiments, the isolated antigen binding protein is a single domain antibody.
在某些实施方式中，所述单域抗体包含HCDR3，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。In certain embodiments, the single domain antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述单域抗体包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。In certain embodiments, the single domain antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.
在某些实施方式中，所述单域抗体包含HCDR1，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S。 In some embodiments, the single domain antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence represented by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein, X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, and X 5 is S.
在某些实施方式中，所述单域抗体的HCDR1包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。In certain embodiments, the HCDR1 of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22.
在某些实施方式中，所述单域抗体包含HCDR1，HCDR2和HCDR3，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。 In certain embodiments, the single domain antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises an amino acid sequence represented by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, Wherein, X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述单域抗体包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。In certain embodiments, the single domain antibody comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22, and the HCDR2 comprises The amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述单域抗体包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:29所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO:29 The amino acid sequence shown.
在某些实施方式中，所述单域抗体包含H-FR1，其中所述H-FR1包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR1, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:9.
在某些实施方式中，所述单域抗体包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:32所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:32 .
在某些实施方式中，所述单域抗体包含H-FR2，所述H-FR2包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:9.
在某些实施方式中，所述单域抗体包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:31所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31 .
在某些实施方式中，所述单域抗体包含H-FR3，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14或SEQ ID NO:18所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR3 comprising amino acids shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:18 sequence.
在某些实施方式中，所述单域抗体包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO:33 The amino acid sequence shown.
在某些实施方式中，所述单域抗体包含H-FR4，所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR4 comprising the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
在某些实施方式中，所述单域抗体包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:29所示的氨基酸序列，所述H-FR2包含SEQ ID NO:32所示的氨基酸序列，所述H-FR3包含SEQ ID NO:31所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 29, the H- FR2 comprises the amino acid sequence shown in SEQ ID NO:32, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:33.
在某些实施方式中，所述单域抗体包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5或SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14或SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises amino acids shown in SEQ ID NO:4 or SEQ ID NO:9 Sequence, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10, and the H-FR3 comprises SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:14 or SEQ ID NO:10 The amino acid sequence shown in ID NO:18, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
在某些实施方式中，所述单域抗体包含选自下述任一组H-FR1，H-FR2，H-FR3和H-FR4；1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的 氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；2)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；3)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；4)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；5)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；6)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；7)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；8)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:27所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列。In certain embodiments, the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups; 1) the H-FR1 comprises SEQ ID NO:4 The amino acid sequence shown, the H-FR2 includes the amino acid sequence shown in SEQ ID NO:5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 7; 2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:10 The amino acid sequence shown in ID NO:11, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 3) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the H -FR2 comprises the amino acid sequence shown in SEQ ID NO:10, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 4 ) said H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 5) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 The amino acid sequence shown, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 6) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:12; The amino acid sequence shown in ID NO:9, the H-FR2 includes the amino acid sequence shown in SEQ ID NO:10, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:18, and the H-FR4 Comprising the amino acid sequence shown in SEQ ID NO: 12; 7) the H-FR1 contains the amino acid sequence shown in SEQ ID NO: 9, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18, and said H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12; 8) said H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9 Sequence, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:27, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:27 Amino acid sequence shown in ID NO:12.
在某些实施方式中，所述单域抗体包含重链可变区VH，所述VH包含SEQ ID NO:34所示的氨基酸序列。In certain embodiments, the single domain antibody comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:34.
在某些实施方式中，所述单域抗体包含重链可变区VH，所述VH包含SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:28所示的氨基酸序列。In certain embodiments, the single domain antibody comprises a heavy chain variable region VH comprising SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID The amino acid sequence shown in NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:28 .
在某些实施方式中，所述单域抗体包含重链恒定区，且所述重链恒定区包含源自IgG的恒定区。In certain embodiments, the single domain antibody comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region.
在某些实施方式中，所述单域抗体的所述重链恒定区包括源自人IgG的恒定区。In certain embodiments, said heavy chain constant region of said single domain antibody comprises a constant region derived from human IgG.
在某些实施方式中，所述单域抗体的所述重链恒定区包含SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37或SEQ ID NO:38所示的氨基酸序列。In certain embodiments, the heavy chain constant region of the single domain antibody comprises the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.
另一方面，本申请提供了一种多肽，其包含本申请所述的分离的抗原结合蛋白。In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein described herein.
另一方面，本申请提供了一种免疫缀合物，其包含本申请所述的分离的抗原结合蛋白或本申请所述的多肽。In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein or the polypeptide described herein.
另一方面，本申请提供了一种分离的核酸分子，所述分离的核酸分子编码本申请所述的分离的抗原结合蛋白，或者本申请所述的多肽。In another aspect, the present application provides an isolated nucleic acid molecule encoding the isolated antigen-binding protein described herein, or the polypeptide described herein.
另一方面，本申请提供了一种载体，其包含本申请所述的分离的核酸分子。In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.
另一方面，本身请提供了一种细胞，其包含本申请所述的分离的抗原结合蛋白，本申请所述的多肽，本申请所述的免疫缀合物，本申请所述的分离的核酸分子和/或本申请所述的载体。In another aspect, please provide a cell comprising the isolated antigen binding protein described in the present application, the polypeptide described in the present application, the immunoconjugate described in the present application, the isolated nucleic acid described in the present application Molecules and/or vectors described herein.
另一方面，本申请提供了一种制备本申请所述的分离的抗原结合蛋白或本申请所述的多肽的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白或本申请所述的多肽表达的条件下，培养本申请所述的细胞。In another aspect, the application provides a method for preparing the isolated antigen-binding protein described in the application or the polypeptide described in the application, the method comprising making the isolated antigen-binding protein described in the application or the polypeptide described in the application The cells described in the present application are cultured under the condition that the polypeptide is expressed.
另一方面，本申请提供了一种药物组合物，其包含本申请所述的分离的抗原结合蛋白，本申请所述的多肽，本申请所述的免疫缀合物，本申请所述的分离的核酸分子，本申请所述的载体，本申请所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein described in the present application, the polypeptide described in the present application, the immunoconjugate described in the present application, the isolated antigen-binding protein described in the present application The nucleic acid molecule of the present application, the carrier described in the present application, the cell described in the present application, and/or the pharmaceutically acceptable adjuvant and/or vehicle.
另一方面，本申请提供了一种药物组合，其包含本申请所述的分离的抗原结合蛋白和免疫检查点抑制剂。In another aspect, the present application provides a pharmaceutical combination comprising the isolated antigen-binding protein described in the present application and an immune checkpoint inhibitor.
在某些实施方式中，所述免疫检查点抑制剂包括抑制PD-1/PD-L1相互作用的物质。In certain embodiments, the immune checkpoint inhibitor includes a substance that inhibits PD-1/PD-L1 interaction.
在某些实施方式中，所述免疫检查点抑制剂选自下组：PD-1/PD-L1阻断剂、PD-1拮抗剂、PD-L1拮抗剂、PD-1抑制剂和PD-L1抑制剂。In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of PD-1/PD-L1 blockers, PD-1 antagonists, PD-L1 antagonists, PD-1 inhibitors, and PD- L1 inhibitor.
在某些实施方式中，所述免疫检测点抑制剂包括抗PD-1抗体。In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.
在某些实施方式中，所述抗PD-1抗体包含HCDR3，所述HCDR3包含SEQ ID NO:39所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:39.
在某些实施方式中，所述抗PD-1抗体包含HCDR2，所述HCDR2包含SEQ ID NO:40所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:40.
在某些实施方式中，所述抗PD-1抗体包含HCDR1，所述HCDR1包含SEQ ID NO:41所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41.
在某些实施方式中，所述抗PD-1抗体包含重链可变区VH，所述VH包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:39所示的氨基酸序列；所述HCDR2包含SEQ ID NO:40所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:41所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 39; the HCDR2 Comprising the amino acid sequence shown in SEQ ID NO:40; and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:41.
在某些实施方式中，所述抗PD-1抗体包含重链可变区VH，所述VH包含SEQ ID NO: 42所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 42.
在某些实施方式中，所述抗PD-1抗体包含LCDR3，所述LCDR3包含SEQ ID NO:43所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:43.
在某些实施方式中，所述抗PD-1抗体包含LCDR2，所述LCDR2包含SEQ ID NO:44所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO:44.
在某些实施方式中，所述抗PD-1抗体包含LCDR1，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:45.
在某些实施方式中，所述抗PD-1抗体包含轻链可变区VL，所述VL包含LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:43所示的氨基酸序列；所述LCDR2包含SEQ ID NO:44所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:45所示的氨基酸序列。In some embodiments, the anti-PD-1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 43; the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:44; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:45.
在某些实施方式中，所述抗PD-1抗体包含轻链可变区VL，所述VL包含SEQ ID NO:46所示的氨基酸序列。In certain embodiments, the anti-PD-1 antibody comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:46.
在某些实施方式中，所述抗PD-1抗体包括帕博丽珠单抗。In certain embodiments, the anti-PD-1 antibody comprises pembrolizumab.
在某些实施方式中，所述免疫检查点抑制剂包括抗PD-L1抗体。In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.
在某些实施方式中，所述抗PD-L1抗体包含HCDR3，所述HCDR3包含SEQ ID NO:47所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:47.
在某些实施方式中，所述抗PD-L1抗体包含HCDR2，所述HCDR2包含SEQ ID NO:48所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:48.
在某些实施方式中，所述抗PD-L1抗体包含HCDR1，所述HCDR1包含SEQ ID NO:49所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:49.
在某些实施方式中，所述抗PD-L1抗体包含重链可变区VH，所述VH包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:47所示的氨基酸序列；所述HCDR2包含SEQ ID NO:48所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:49所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 47; the HCDR2 Comprising the amino acid sequence shown in SEQ ID NO:48; and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:49.
在某些实施方式中，所述抗PD-L1抗体包含重链可变区VH，所述VH包含SEQ ID NO:50所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:50.
在某些实施方式中，所述抗PD-L1抗体包含LCDR3，所述LCDR3包含SEQ ID NO:51所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:51.
在某些实施方式中，所述抗PD-L1抗体包含LCDR2，所述LCDR2包含SEQ ID NO:52所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:52.
在某些实施方式中，所述抗PD-L1抗体包含LCDR1，所述LCDR1包含SEQ ID NO:53 所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53.
在某些实施方式中，所述抗PD-L1抗体包含轻链可变区VL，所述VL包含LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:51所示的氨基酸序列；所述LCDR2包含SEQ ID NO:52所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:53所示的氨基酸序列。In some embodiments, the anti-PD-L1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 51; the LCDR2 comprising the amino acid sequence shown in SEQ ID NO:52; and the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53.
在某些实施方式中，所述抗PD-L1抗体包含轻链可变区VL，所述VL包含SEQ ID NO:54所示的氨基酸序列。In certain embodiments, the anti-PD-L1 antibody comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:54.
在某些实施方式中，所述抗PD-L1抗体包括阿替利珠单抗。In certain embodiments, the anti-PD-L1 antibody comprises atezolizumab.
另一方面，本申请提供了一种用于检测或测定Siglec15的方法，所述方法包括使用本申请所述的分离的抗原结合蛋白或本申请所述的多肽。In another aspect, the present application provides a method for detecting or measuring Siglec15, the method comprising using the isolated antigen-binding protein described in the present application or the polypeptide described in the present application.
另一方面，本申请提供了一种Siglec15的检测试剂盒，其包含本申请所述的分离的抗原结合蛋白或本申请所述的多肽。In another aspect, the present application provides a Siglec15 detection kit, which comprises the isolated antigen-binding protein or the polypeptide described in the present application.
另一方面，本身请提供了一种所述的分离的抗原结合蛋白或所述的多肽在制备试剂盒中的用途。In another aspect, please provide a use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit.
另一方面，本申请提供了一种调节免疫应答的方法，其包括向有需要的受试者施用有效量的本申请所述的分离的抗原结合蛋白，本申请所述的多肽，本申请所述的免疫缀合物，本申请所述的分离的核酸分子，本申请所述的载体，本申请所述的细胞，本申请所述的药物组合物和/或本申请所述的药物组合，和/或药学上可接受的治疗剂。On the other hand, the present application provides a method for regulating immune response, which comprises administering to a subject in need an effective amount of the isolated antigen-binding protein described in the present application, the polypeptide described in the present application, the polypeptide described in the present application, The immunoconjugate described herein, the isolated nucleic acid molecule described herein, the carrier described herein, the cell described herein, the pharmaceutical composition described herein and/or the pharmaceutical combination described herein, and/or a pharmaceutically acceptable therapeutic agent.
另一方面，本申请提供了一种本申请所述的分离的抗原结合蛋白，本申请所述的多肽，本申请所述的免疫缀合物，本申请所述的分离的核酸分子，本申请所述的载体，本申请所述的细胞，本申请所述的药物组合物和/或本申请所述的药物组合在制备预防和/或治疗疾病或病症的药物中的用途。In another aspect, the application provides an isolated antigen-binding protein described in the application, the polypeptide described in the application, the immunoconjugate described in the application, the isolated nucleic acid molecule described in the application, the polypeptide described in the application Use of the carrier, the cell described in the application, the pharmaceutical composition described in the application and/or the combination of drugs described in the application in the preparation of drugs for preventing and/or treating diseases or diseases.
在某些实施方式中，在本申请所述的制备预防和/或治疗疾病或病症的药物中的用途中，所述疾病或病症包括肿瘤。In certain embodiments, in the use of the medicament for preventing and/or treating a disease or disorder described in the present application, the disease or disorder includes a tumor.
在某些实施方式中，在本申请所述的制备预防和/或治疗疾病或病症的药物中的用途中，所述肿瘤包括与Siglec15的表达相关的肿瘤。In certain embodiments, in the application of the medicament for preventing and/or treating diseases or conditions described in this application, the tumor includes a tumor associated with the expression of Siglec15.
在某些实施方式中，在本申请所述的制备预防和/或治疗疾病或病症的药物中的用途中，所述肿瘤包括实体瘤。In some embodiments, in the use of the medicament for preventing and/or treating a disease or condition described in this application, the tumor includes a solid tumor.
在某些实施方式中，在本申请所述的制备预防和/或治疗疾病或病症的药物中的用途中，所述肿瘤包括结肠癌。In some embodiments, in the use of the medicament for preventing and/or treating diseases or conditions described in this application, the tumor includes colon cancer.
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.
附图说明Description of drawings
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:
图1显示的是本申请所述Siglec15结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合曲线。Figure 1 shows the binding curve of the Siglec15-binding protein described in the present application to human Siglec15 expressed on the surface of CHOK1 cells.
图2A显示的是本申请所述Siglec15抗原结合蛋白促进CD8 +T细胞的增殖。 Figure 2A shows that the Siglec15 antigen-binding protein described in this application promotes the proliferation of CD8 + T cells.
图2B显示的是本申请所述Siglec15抗原结合蛋白促进CD4 +T细胞的增殖。 Figure 2B shows that the Siglec15 antigen-binding protein described in this application promotes the proliferation of CD4 + T cells.
图2C显示的是本申请所述Siglec15抗原结合蛋白促进培养基上清中IFN-γ的表达。Figure 2C shows that the Siglec15 antigen-binding protein described in this application promotes the expression of IFN-γ in the culture supernatant.
图3A-B显示的是本申请所述人源化Siglec15结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合曲线。3A-B show the binding curves of the humanized Siglec15-binding protein described in the present application and human Siglec15 expressed on the surface of CHOK1 cells.
图4A显示的是本申请所述人源化Siglec15抗原结合蛋白促进CD8 +T细胞的增殖。 Figure 4A shows that the humanized Siglec15 antigen-binding protein described in this application promotes the proliferation of CD8 + T cells.
图4B显示的是本申请所述人源化Siglec15抗原结合蛋白促进CD4 +T细胞的增殖。 Figure 4B shows that the humanized Siglec15 antigen-binding protein described in this application promotes the proliferation of CD4 + T cells.
图4C显示的是本申请所述人源化Siglec15抗原结合蛋白促进培养基上清中IFN-γ的表达。Figure 4C shows that the humanized Siglec15 antigen-binding protein of the present application promotes the expression of IFN-γ in the culture supernatant.
图5A-B显示的是本申请所述人源化Siglec15抗原结合蛋白与人Siglec15抗原的结合曲线。5A-B show the binding curves of the humanized Siglec15 antigen-binding protein described in the present application to human Siglec15 antigen.
图5C-D显示的是本申请所述人源化Siglec15抗原结合蛋白与猴Siglec15抗原的结合曲线。5C-D show the binding curves of the humanized Siglec15 antigen-binding protein described in the present application to the monkey Siglec15 antigen.
图5E-F显示的是本申请所述人源化Siglec15抗原结合蛋白与鼠Siglec15抗原的结合曲线。5E-F show the binding curves of the humanized Siglec15 antigen-binding protein described in the present application to the mouse Siglec15 antigen.
图6A显示的是本申请所述抗原结合蛋白在野生型C57BL/6N小鼠体内的抗肿瘤药效。Figure 6A shows the anti-tumor efficacy of the antigen-binding protein described in this application in wild-type C57BL/6N mice.
图6B显示的是本申请所述抗原结合蛋白施用后，野生型C57BL/6N小鼠的体重变化。Fig. 6B shows the body weight changes of wild-type C57BL/6N mice after administration of the antigen-binding protein described in the present application.
图7A显示的是本申请所述抗原结合蛋白在人Siglec15转基因小鼠体内的抗肿瘤药效。Figure 7A shows the anti-tumor efficacy of the antigen-binding protein described in this application in human Siglec15 transgenic mice.
图7B显示的是本申请所述抗原结合蛋白施用后，人Siglec15转基因小鼠的体重变化。Fig. 7B shows the body weight changes of human Siglec15 transgenic mice after administration of the antigen-binding protein described in the present application.
图8A显示的是本申请所述抗原结合蛋白在人Siglec15转基因小鼠体内的抗肿瘤药效。Figure 8A shows the anti-tumor efficacy of the antigen-binding protein described in this application in human Siglec15 transgenic mice.
图8B显示的是本申请所述抗原结合蛋白施用后，人Siglec15转基因小鼠的体重变化。Fig. 8B shows the body weight changes of human Siglec15 transgenic mice after administration of the antigen-binding protein described in the present application.
图9显示的是本申请所述人源化Siglec15抗原结合蛋白抑制破骨细胞的形成。Figure 9 shows that the humanized Siglec15 antigen-binding protein described in the present application inhibits the formation of osteoclasts.
图10A显示的是本申请所述抗原结合蛋白Ab5、Ab10和PD-1抗体联用在人PD-1/PD-L1转基因小鼠体内的抗肿瘤药效。Figure 10A shows the anti-tumor efficacy of the combination of antigen-binding proteins Ab5, Ab10 and PD-1 antibody described in this application in human PD-1/PD-L1 transgenic mice.
图10B显示的是本申请所述抗原结合蛋白Ab5、Ab10和PD-1抗体联用后，人PD-1/PD-L1转基因小鼠的体重变化。Figure 10B shows the body weight changes of human PD-1/PD-L1 transgenic mice after the combination of antigen binding proteins Ab5, Ab10 and PD-1 antibody described in this application.
图11A显示的是本申请所述抗原结合蛋白Ab5、Ab10和PD-L1抗体联用在人PD-1/PD-L1转基因小鼠体内的抗肿瘤药效。Figure 11A shows the anti-tumor efficacy of the combined use of antigen-binding proteins Ab5, Ab10 and PD-L1 antibodies described in this application in human PD-1/PD-L1 transgenic mice.
图11B显示的是本申请所述抗原结合蛋白Ab5、Ab10和PD-L1抗体联用后，人PD-1/PD-L1转基因小鼠的体重变化。Figure 11B shows the body weight changes of human PD-1/PD-L1 transgenic mice after the combination of antigen binding proteins Ab5, Ab10 and PD-L1 antibody described in this application.
具体实施方式Detailed ways
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。The implementation of the invention of the present application will be described by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.
术语定义Definition of Terms
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
在本申请中，术语“Siglec15蛋白”，“Siglec-15”或“Siglec15抗原”可以互换使用，并且包括Siglec15的任何功能活性片段、变体和同源物，其由细胞天然表达或在用Siglec15基因转染的细胞上表达。本申请所述Siglec15可以包括Siglec15抗原或其片段或其抗原决定簇。本申请所述Siglec15可以包括人Siglec15、鼠Siglec15和/或猴Siglec15。例如，所述人Siglec15为人源Siglec15，在NCBI中，编码其的核苷酸序列编号可以是284266，其在UniProt/Swiss-Prot中的登录号可以为Q6ZMC9。例如，所述鼠Siglec15为小鼠Siglec15，在NCBI中，编码其的核苷酸序列编号可以是620235。例如，所述猴Siglec15为食蟹猴Siglec15，在NCBI中，编码其的核苷酸序列编号可以是700656。In this application, the terms "Siglec15 protein", "Siglec-15" or "Siglec15 antigen" are used interchangeably and include any functionally active fragments, variants and homologues of Siglec15 that are naturally expressed by cells or used in Expression on Siglec15 gene transfected cells. The Siglec15 described in the present application may include Siglec15 antigen or a fragment thereof or an antigenic determinant thereof. The Siglec15 described in the present application may include human Siglec15, mouse Siglec15 and/or monkey Siglec15. For example, the human Siglec15 is human Siglec15. In NCBI, the nucleotide sequence number encoding it may be 284266, and its accession number in UniProt/Swiss-Prot may be Q6ZMC9. For example, the mouse Siglec15 is mouse Siglec15, and the nucleotide sequence code encoding it may be 620235 in NCBI. For example, the monkey Siglec15 is cynomolgus monkey Siglec15, and the nucleotide sequence code encoding it may be 700656 in NCBI.
在本申请中，术语“功能活性片段”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的功能活性片段是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得编码功能活性片段的序列，只要保持原来的功能活性即可。In the present application, the term "functionally active fragment" refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence. In the context of this application, a functionally active fragment of any given sequence is one in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least A sequence of endogenous function. Sequences encoding functionally active fragments can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in naturally occurring proteins and/or polynucleotides , as long as the original functional activity is maintained.
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。In the present application, the term "derivative" generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and and/or added, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。In this application, the term "analogue" generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。Typically, amino acid substitutions, e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.
在本申请中，术语“同源物”通常是指与天然存在序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。In the present application, the term "homologue" generally refers to an amino acid sequence or nucleotide sequence having a certain homology to a naturally occurring sequence. The term "homology" may be equated with sequence "identity". Homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence . Typically, a homologue will comprise the same active site, etc., as the subject amino acid sequence. Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity. In this application, a sequence having a percentage identity of any one of the SEQ ID NOs of the mentioned amino acid sequence or nucleotide sequence means having said percentage identity over the entire length of the mentioned SEQ ID NO the sequence of. To determine sequence identity, sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、***或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含 具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。The proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid. Paragine, Glutamine, Serine, Threonine and Tyrosine.
在本申请中，术语“抗原结合蛋白”通常是指能与抗原或其片段结合的抗体或其片段。所述抗原结合蛋白可以包括人源化抗体、嵌合抗体、重组抗体、单链抗体、单克隆抗体、多克隆抗体、Fab片段、F(ab′)2片段、IgD抗体、IgE抗体、IgM抗体、IgG1抗体、IgG2抗体、IgG3抗体和/或IgG4抗体，以及它们的片段。本申请所述抗原结合蛋白可以包括来源于人、来源于鼠、来源于猴和/或来源于羊驼的抗原结合蛋白。本申请所述抗原结合蛋白可以包括人源化的Siglec15单域抗体。In this application, the term "antigen-binding protein" generally refers to an antibody or fragment thereof capable of binding to an antigen or fragment thereof. The antigen binding protein may include humanized antibody, chimeric antibody, recombinant antibody, single chain antibody, monoclonal antibody, polyclonal antibody, Fab fragment, F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody , IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and/or IgG4 antibodies, and fragments thereof. The antigen-binding proteins described in the present application may include antigen-binding proteins derived from humans, mice, monkeys and/or alpacas. The antigen binding protein described in the present application may comprise a humanized Siglec15 single domain antibody.
在本申请中，术语“特异性结合”通常是指结合蛋白(例如抗原结合蛋白)特异地识别一种配体(例如抗原)并与其发生结合的相互作用。本申请所述特异性结合可以包括至少一种物种来源的抗原结合蛋白、抗原结合蛋白片段和/或多肽与至少一种物种来源的抗原、抗原决定簇、抗原表位，由于特定的空间构象而识别并结合。例如，本申请所述特异性结合可以包括本申请所述抗原结合蛋白特异性的识别Siglec15并阻断Siglec15的功能。In the present application, the term "specific binding" generally refers to an interaction in which a binding protein (such as an antigen-binding protein) specifically recognizes a ligand (such as an antigen) and binds to it. The specific binding described in the present application may include at least one species-derived antigen-binding protein, antigen-binding protein fragment and/or polypeptide and at least one species-derived antigen, antigenic determinant, antigenic epitope, due to a specific spatial conformation Identify and combine. For example, the specific binding described in the present application may include that the antigen binding protein described in the present application specifically recognizes Siglec15 and blocks the function of Siglec15.
在本申请中，术语“K D”(同样地，“K D”或“K D”)通常指“亲和常数”或“平衡解离常数”，并指在滴定测量中在平衡时、或者通过将解离速率常数(kd)除以结合速率常数(ka)所获得的值。使用结合速率常数(ka)、解离速率常数(kd)和平衡解离常数(K D)表示结合蛋白(例如本申请所述的分离的抗原结合蛋白)对抗原(例如，Siglec15蛋白)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术提供了高灵敏度以及在生理缓冲液中在平衡时检查样品的能力。例如，可以通过Biacore(生物分子相互作用分析)测定(例如，可以从BIAcoreInternationalAB，aGEHealthcarecompany，Uppsala，瑞典获得的仪器)所述K D值，也可以使用其他实验途径和仪器例如Octet检测。另外，也可以使用可以从SapidyneInstruments(Boise，Idaho)获得的KinExA(动态排阻测定(KineticExclusionAssay))测定所述K D值，或者使用表面等离子共振仪(SPR)测定所述K D值。例如，也可以通过胺偶联试剂盒测定所述K D值。 In this application, the term "K D " (likewise, "K D " or "K D ") generally refers to "affinity constant" or "equilibrium dissociation constant" and refers to a titration measurement at equilibrium, or Value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka). Binding of a binding protein (e.g., an isolated antigen-binding protein described herein) to an antigen (e.g., Siglec15 protein) is expressed using an on-rate constant (ka), an off-rate constant (kd), and an equilibrium dissociation constant ( KD ) affinity. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the KD value can be determined by Biacore (Biomolecular Interaction Analysis) (for example, an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden), and other experimental approaches and instruments such as Octet can also be used. In addition, the KD value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho), or using a surface plasmon resonance (SPR) instrument. For example, the KD value can also be determined by an amine coupling kit.
在本申请中，术语“增殖抑制”通常是指使细胞***(细胞的对称或不对称***)或细胞生长停滞或减缓的过程。本申请所述“增殖抑制”可以指在肿瘤微环境中，Siglec15可以抑制T细胞的活化、生长和/或***。In this application, the term "proliferation inhibition" generally refers to the process of arresting or slowing cell division (symmetric or asymmetric division of cells) or cell growth. The "proliferation inhibition" mentioned in this application may mean that Siglec15 can inhibit the activation, growth and/or division of T cells in the tumor microenvironment.
在本申请中，术语“抗体”通常是指一类能与特定抗原、蛋白、片段或多肽发生特异性结合并发挥功能的免疫球蛋白。所述抗体可以包括：重链恒定区、轻链恒定区、重链可变区和/或轻链可变区。所述可变区可以包括分离的CDR区和分离的FR区。所述恒定区可以包 括分离的CH1结构域、分离的CH2结构域和/或分离的CH3结构域。本申请所述抗体可以包括整个抗体、任何结合片段和/或其单链。例如，所述任何结合片段可以包括：Fab、Fab’、F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。本申请所述的抗体可以包括单克隆抗体、多克隆抗体、单链抗体、单域抗体、重组抗体、嵌合抗体、人源化抗体和/或全人源抗体。本申请所述抗体可以包括来源于人、来源于鼠、来源于猴和/或来源于羊驼的抗体。 In this application, the term "antibody" generally refers to a class of immunoglobulins that can specifically bind to a specific antigen, protein, fragment or polypeptide and exert functions. The antibody may comprise: a heavy chain constant region, a light chain constant region, a heavy chain variable region and/or a light chain variable region. The variable region may comprise separate CDR regions and separate FR regions. The constant region may comprise an isolated CH1 domain, an isolated CH2 domain and/or an isolated CH3 domain. Antibodies described herein may include whole antibodies, any binding fragments and/or single chains thereof. For example, any of the binding fragments may include: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb. The antibodies described herein may include monoclonal antibodies, polyclonal antibodies, single chain antibodies, single domain antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies. The antibodies described herein may include antibodies derived from humans, mice, monkeys and/or alpacas.
在本申请中，术语“单克隆抗体”通常是指一群基本同源的抗体，即包含该群的各个抗体除了可能的以微量存在的天然发生的突变之外是相同的。单克隆抗体是高度特异性的，直接针对单个抗原性位点。例如，所述单克隆抗体可以通过杂交瘤技术制备或者通过使用重组DNA方法在细菌、真核动物或植物细胞中产生。单克隆抗体也可以得自噬菌体抗体文库，使用例如Clackson etal.,Nature,352:624-628(1991)和Marks et al.,Mol.Biol.，222:581-597(1991)所述的技术进行。In this application, the term "monoclonal antibody" generally refers to a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. For example, the monoclonal antibodies can be produced by hybridoma technology or produced in bacterial, eukaryotic or plant cells by using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991) conduct.
在本申请中，术语“嵌合抗体”通常是指这样的抗体，其中每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别，而该链的其余区段则与另一物种中的相应序列同源。例如，轻链和重链的可变区均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。所述可变区具有易于制备的优点，并且其特异性不受与其组合的恒定区的来源的影响。同时，由于嵌合抗体的恒定区可来源于人类，因此嵌合在注射时抗体引发免疫应答的可能性会低于使用恒定区为非人来源的抗体。In this application, the term "chimeric antibody" generally refers to an antibody in which a part of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class, and The remainder of the chain is then homologous to the corresponding sequence in another species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human) . For example, to obtain chimeric antibodies, B cells or hybridoma cells of non-human origin can be used to produce variable regions combined with constant regions of human origin. The variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region it is combined with. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody whose constant region is of non-human origin.
在本申请中，术语“人源化抗体”通常是指一种嵌合抗体，其含有较少的来自非人免疫球蛋白的序列，从而降低异种抗体引入到人类中时的免疫原性，同时保持抗体的完全抗原结合亲和力和特异性。例如，可以使用CDR移植(Jones et al.,Nature 321:522(1986))及其变体；包括“重塑”(reshaping)，(Verhoeyen,et al.,1988Science 239:1534-1536；Riechmann,et al.,1988Nature 332:323-337；Tempest,et al.,Bio/Technol 1991 9:266-271)，“高度加成”(hyperchimerization)，(Queen,et al.,1989Proc Natl Acad Sci USA 86:10029-10033；Co,et al.,1991 Proc Natl Acad Sci USA 88:2869-2873；Co,et al.,1992J Immunol 148:1149-1154)和“贴面”(veneering)，(Mark,et al.,“Derivation of therapeutically active humanized and veneered anti-CD18antibodies.”In:Metcalf B W,Dalton B J,eds.Cellular adhesion:molecular definition to therapeutic potential.New York:Plenum Press,1994:291-312)、表面重建(美国专利US5639641) 等技术手段，对非人源的结合域进行人源化。如果其他区域，例如铰链区和恒定区结构域也源自非人来源，则这些区域也可以被人源化。In this application, the term "humanized antibody" generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing the immunogenicity of a heterologous antibody when introduced into a human, while simultaneously Preserves the full antigen-binding affinity and specificity of the antibody. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof; including "reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), "high addition" (hyperchimerization), (Queen, et al., 1989Proc Natl Acad Sci USA 86 : 10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co, et al., 1992J Immunol 148: 1149-1154) and "veneering" (veneering), (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), Surface reconstruction (US Patent US5639641) and other technical means are used to humanize the non-human binding domain. Other regions, such as the hinge and constant region domains, can also be humanized if they are also derived from non-human sources.
在本申请中，术语“单域抗体”通常是指天然缺失轻链的纳米抗体。本申请所述单域抗体可以包括重链可变区和常规的重链CH2、CH3区。本申请所述的单域抗体可以是抗原结合蛋白的最小结合单元。本申请所述单域抗体可以包括Fab、Fab’、F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。 In this application, the term "single domain antibody" generally refers to Nanobodies that are naturally devoid of light chains. The single domain antibody described in this application may include heavy chain variable regions and conventional heavy chain CH2 and CH3 regions. The single domain antibody described in the present application can be the smallest binding unit of an antigen binding protein. The single domain antibody described herein may comprise Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
在本申请中，术语“抗原结合片段”通常是指抗体中发挥特异性结合抗原功能的一个或多个片段。抗体的抗原结合功能可通过抗体的全长片段来实现。抗体的抗原结合功能也可通过以下来实现：包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的重链，或者，包括Fv、scFv、dsFv、Fab、Fab’或F(ab’)2的片段的轻链。(1)Fab片段，通常为由VL、VH、CL和CH结构域组成的一价片段；(2)F(ab’)2片段，包含通过铰链区处的二硫键连接的两个Fab片段的二价片段；(3)由VH和CH结构域组成的Fd片段；(4)由抗体单臂的VL和VH结构域组成的Fv片段；(5)由VH结构域组成的dAb片段(Ward等，(1989)Nature341：544-546)；(6)分离的互补决定区(CDR)和(7)可任选地通过接头连接的两个或以上分离的CDR的组合。例如，还可包括由VL和VH配对形成的一价单链分子Fv(scFv)(参见Bird等(1988)Science 242：423-426；以及Huston等(1988)Proc.Natl.Acad.Sci.85：5879-5883)。例如，还可包括缺失抗体轻链而只有重链可变区的一类抗体VHH(例如，可参见康晓圳等，生物工程学报,2018,34(12):1974-1984)。所述“抗原结合部分”还可包括免疫球蛋白融合蛋白，所述融合蛋白包含选自以下的结合结构域：(1)与免疫球蛋白铰链区多肽融合的结合结构域多肽；(2)与铰链区融合的免疫球蛋白重链CH2恒定区；和(3)与CH2恒定区融合的免疫球蛋白重链CH3恒定区。In this application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of antibodies can be realized by full-length fragments of antibodies. The antigen-binding function of an antibody can also be achieved by comprising a heavy chain of a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab'), or by comprising a Fv, scFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2. (1) Fab fragment, usually a monovalent fragment consisting of VL, VH, CL and CH domains; (2) F(ab')2 fragment, comprising two Fab fragments linked by a disulfide bond at the hinge region (3) Fd fragment composed of VH and CH domains; (4) Fv fragment composed of VL and VH domains of antibody single arm; (5) dAb fragment composed of VH domains (Ward et al., (1989) Nature 341 :544-546); (6) an isolated complementarity determining region (CDR) and (7) a combination of two or more isolated CDRs optionally linked by a linker. For example, the monovalent single-chain molecule Fv (scFv) formed by the pairing of VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl.Acad.Sci.85 :5879-5883). For example, a type of antibody VHH that lacks the light chain of the antibody and only has the variable region of the heavy chain can also be included (for example, see Kang Xiaozhen et al., Acta Biological Engineering, 2018, 34(12): 1974-1984). The "antigen binding portion" may also include an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
在本申请中，术语“重链恒定区”或“CH”通常是指免疫球蛋白重链结构中分离的CH1结构域、分离的CH2结构域和/或分离的CH3结构域。本申请所述重链恒定区可以包括在单域抗体的结构中与重链可变区VH片段C端相连的片段。本申请所述重链恒定区可以包含源自IgG的恒定区。In the present application, the term "heavy chain constant region" or "CH" generally refers to an isolated CH1 domain, an isolated CH2 domain and/or an isolated CH3 domain in an immunoglobulin heavy chain structure. The heavy chain constant region described in the present application may include a fragment connected to the C-terminus of the VH fragment of the heavy chain variable region in the structure of the single domain antibody. The heavy chain constant region described herein may comprise an IgG-derived constant region.
在本申请中，术语”重链可变区“或”VH“通常是指免疫球蛋白重链结构中包含重链互补决定区CDR1、CDR2、CDR3和框架区FR1、FR2、FR3、FR4的区域。所述重链可变区含有与抗原相互作用的结合结构域。本申请所述重链可变区可以包括在单域抗体结构中与Siglec15特异性结合的区域。本申请所述重链可变区可以包括单域抗体的VHH。In this application, the term "heavy chain variable region" or "VH" generally refers to the region of the immunoglobulin heavy chain structure comprising the heavy chain complementarity determining regions CDR1, CDR2, CDR3 and the framework regions FR1, FR2, FR3, FR4 . The heavy chain variable region contains a binding domain that interacts with the antigen. The heavy chain variable region described in the present application may include a region that specifically binds to Siglec15 in the single domain antibody structure. The heavy chain variable region described herein may comprise the VHH of a single domain antibody.
在本申请中，术语“编码”通常是指核酸(例如脱氧核糖核酸)转录互补核酸的特性，包括能翻译成多肽的核酸。例如，脱氧核糖核酸可编码从脱氧核糖核酸转录的RNA。脱氧核糖核酸可类似地编码从脱氧核糖核酸所转录的RNA翻译的多肽。In this application, the term "encoding" generally refers to the property of a nucleic acid (such as deoxyribonucleic acid) transcribing a complementary nucleic acid, including nucleic acid capable of being translated into a polypeptide. For example, deoxyribonucleic acid can encode RNA transcribed from deoxyribonucleic acid. A deoxyribonucleic acid may similarly encode a polypeptide translated from RNA transcribed from the deoxyribonucleic acid.
在本申请中，术语“IgG”通常是指一类免疫球蛋白γ基因编码的抗体类别的多肽。在人类中，此类别包括IgG1、IgG2、IgG3和IgG4。在小鼠中，此类别包括IgG1、IgG2a、IgG2b和IgG3。本申请所述IgG包括重链恒定区、轻链恒定区、重链可变区和/或轻链可变区。本申请所述IgG的恒定区可以为人源化单域抗体提供重链恒定区结构。In this application, the term "IgG" generally refers to a class of polypeptides of the antibody class encoded by immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3 and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b, and IgG3. The IgG described herein includes a heavy chain constant region, a light chain constant region, a heavy chain variable region and/or a light chain variable region. The IgG constant region described herein can provide a heavy chain constant region structure for a humanized single domain antibody.
在本申请中，术语“免疫缀合物”通常是指与抗原结合蛋白或其片段相连接的其他组分。所述免疫缀合物可以包括与抗原结合蛋白融合或偶联的其他的多肽、治疗剂、探针和/或其他抗体。In this application, the term "immunoconjugate" generally refers to other components linked to an antigen-binding protein or fragment thereof. The immunoconjugates may include other polypeptides, therapeutic agents, probes and/or other antibodies fused or conjugated to the antigen binding protein.
在本申请中，术语“核酸分子”通常是指包括脱氧核糖核酸(DNA)和核糖核酸(RNA)的，由许多核苷酸单体聚合成的生物大分子化合物。所述核酸分子可以是单链或双链。本申请所述核酸分子可以是编码本申请所涉及的抗体或肽的任意核酸分子。本申请所述核酸分子可以存在于细胞中、载体中和/或以游离形式存在于水或溶液中。所述核酸分子可以包括天然存在的或人工合成的核酸分子。In this application, the term "nucleic acid molecule" generally refers to a biomacromolecular compound composed of many nucleotide monomers, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid molecule can be single-stranded or double-stranded. The nucleic acid molecule described in the present application may be any nucleic acid molecule encoding the antibody or peptide involved in the present application. The nucleic acid molecules described herein may be present in cells, in vectors and/or in free form in water or solutions. The nucleic acid molecules may include naturally occurring or synthetic nucleic acid molecules.
在本申请中，术语“载体”通常是指在基因工程重组DNA技术中将DNA片段转移至受体细胞的一种能自我复制的DNA分子。所述载体可以包括细菌质粒载体、噬菌体载体和/或动植物病毒载体。本申请所述载体可以是环状DNA载体，包括复制起始位点、多克隆位点、开放阅读框和/或标记基因。In this application, the term "vector" generally refers to a self-replicating DNA molecule that transfers DNA fragments to recipient cells in genetic engineering recombinant DNA technology. The vectors may include bacterial plasmid vectors, phage vectors and/or animal and plant viral vectors. The vector described in this application may be a circular DNA vector, including an origin of replication, a multiple cloning site, an open reading frame and/or a marker gene.
在本申请中，所述“氨基酸序列”通常是指至少一个的氨基酸相互连接形成的顺序。所述氨基酸序列可以是编码多肽或蛋白的主要结构。所述氨基酸序列中氨基酸可以以脱水缩合的形式连接。本申请所述氨基酸序列可以编码多种抗原结合蛋白或其片段。In the present application, the "amino acid sequence" generally refers to the sequence formed by linking at least one amino acid to each other. The amino acid sequence may encode the main structure of a polypeptide or protein. Amino acids in the amino acid sequence can be linked in the form of dehydration condensation. The amino acid sequences described herein may encode various antigen binding proteins or fragments thereof.
在本申请中，术语“多肽”、“肽”、“蛋白质”、“蛋白质片段”可以互换使用，通常是指由一条或多条由氨基酸连接在一起组成的化合物。所述多肽可以包括由至少一个氨基酸与其他氨基酸脱水缩合形成的化合物。本申请所述多肽可以包括抗原结合蛋白及其片段。本申请所述多肽可以包括天然的氨基酸聚合物和人工合成的氨基酸聚合物。In this application, the terms "polypeptide", "peptide", "protein", and "protein fragment" can be used interchangeably, and generally refer to a compound composed of one or more amino acids linked together. The polypeptide may include a compound formed by dehydration condensation of at least one amino acid with other amino acids. Polypeptides described herein may include antigen binding proteins and fragments thereof. The polypeptides described herein may include natural amino acid polymers and artificially synthesized amino acid polymers.
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的多肽或本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述 的多肽或抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方式中，所述细胞可以是免疫细胞。例如，所述免疫细胞可以选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。In this application, the term "cell" generally refers to an individual cell that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing a polypeptide described herein or an antigen binding protein described herein , cell line or cell culture. The cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the polypeptide or antigen-binding protein described in this application. The cells can be obtained by transfecting cells in vitro with the vectors described in this application. The cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells). In some embodiments, the cells can be immune cells. For example, the immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.
在本申请中，术语“检测试剂盒”通常是指包含一组用于检测包括蛋白和/或核酸分子的化学试剂和/或生物大分子的集合。In this application, the term "detection kit" generally refers to a set of chemical reagents and/or biomacromolecules for detection of proteins and/or nucleic acid molecules.
在本申请中，术语“免疫缀合物”通常是指所述其他治疗剂与所述分离的抗原结合蛋白缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述分离的抗原结合蛋白与靶细胞上的抗原的特异性结合，将所述其他治疗剂递送至靶细胞(例如，肿瘤细胞)。此外，所述抗原也可以由所述靶细胞分泌，并位于所述靶细胞外的间隙。In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (for example, covalently linking through a linker molecule) the other therapeutic agent to the isolated antigen-binding protein, the conjugate The other therapeutic agent can be delivered to a target cell (eg, a tumor cell) by specific binding of the isolated antigen binding protein to an antigen on the target cell. In addition, the antigen may also be secreted by the target cell and located in the space outside the target cell.
在本申请中，术语“药物组合物”通常是指包括一种或多种具有药物的生物活性的组合物。本申请所述的药物组合物可以包括本申请所述的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。In this application, the term "pharmaceutical composition" generally refers to a composition comprising one or more biological activities of a drug. The pharmaceutical composition described in the application can comprise the antigen-binding protein described in the application, the nucleic acid molecule described in the application, the vector described in the application and/or the cell described in the application, and optionally pharmaceutically acceptable Accepted adjuvants.
在本申请中，术语“肿瘤”通常是指局部组织细胞增生所形成的赘生物。例如，所述肿瘤可包括实体瘤。例如，所述肿瘤可包括与Siglec15的表达相关的肿瘤。术语“与Siglec15的表达相关的肿瘤”通常是指存在Siglec15表达的肿瘤。所述与Siglec15的蛋白表达相关的肿瘤可以是Siglec15阳性的肿瘤。在Siglec15阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的Siglec15的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。In this application, the term "tumor" generally refers to a neoplasm formed by the proliferation of local tissue cells. For example, the tumor can include a solid tumor. For example, the tumor can include a tumor associated with expression of Siglec15. The term "tumor associated with expression of Siglec15" generally refers to a tumor in which Siglec15 expression is present. The tumor associated with Siglec15 protein expression may be a Siglec15 positive tumor. In Siglec15-positive tumors, compared with normal cells, the protein expression of Siglec15 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
在本申请中，术语“实体瘤”通常是指可通过临床检测(例如，X线射片，CT扫描，B超或触诊)到的有形肿块。例如，所述实体瘤可以包括结肠癌。In this application, the term "solid tumor" generally refers to a tangible mass that can be detected clinically (eg, X-ray film, CT scan, B-ultrasound or palpation). For example, the solid tumor can include colon cancer.
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。In this application, the term "and/or" should be understood as meaning any one of the optional items or both of the optional items.
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".
发明详述Detailed description of the invention
分离的抗原结合蛋白isolated antigen binding protein
一方面，本申请提供一种分离的抗原结合蛋白。所述分离的抗原结合蛋白在Biacore检测中，可以以约5E-09M或以下的K D值(例如，所述K D值为不高于约5E-09M、不高于约4.9E-09M、不高于约4.8E-09M、不高于约4.7E-09M、不高于约4.6E-09M、不高于约4.5E-09M、不高于约4.4E-09M、不高于约4.3E-09M、不高于约4.2E-09M、不高于约4.1E-09M、不高于约4E-09M、不高于约3.0E-09M、不高于约2.5E-09M、不高于约2.0E-09M、不高于约1.5E-09M、不高于约1.0E-09M、不高于约9E-10M、不高于约8E-10M、不高于约5E-11M、不高于约1E-11M或不高于5E-12M或以下)与人Siglec15蛋白特异性结合。 In one aspect, the application provides an isolated antigen binding protein. The isolated antigen-binding protein may have a KD value of about 5E-09M or less in a Biacore assay (for example, the KD value is not higher than about 5E-09M, not higher than about 4.9E-09M, Not higher than about 4.8E-09M, not higher than about 4.7E-09M, not higher than about 4.6E-09M, not higher than about 4.5E-09M, not higher than about 4.4E-09M, not higher than about 4.3 E-09M, not higher than about 4.2E-09M, not higher than about 4.1E-09M, not higher than about 4E-09M, not higher than about 3.0E-09M, not higher than about 2.5E-09M, not higher At about 2.0E-09M, not higher than about 1.5E-09M, not higher than about 1.0E-09M, not higher than about 9E-10M, not higher than about 8E-10M, not higher than about 5E-11M, not higher than Higher than about 1E-11M or not higher than 5E-12M or lower) specifically binds to human Siglec15 protein.
在本申请中，所述分离的抗原结合蛋白在流式检测中，可以以约0.2μg/ml或以下的EC 50值(例如，所述EC 50值为不高于约0.2μg/ml、不高于约0.15μg/ml、不高于约0.13μg/ml、不高于约0.12μg/ml、不高于约0.1μg/ml、不高于约0.09μg/ml、不高于约0.08μg/ml、不高于约0.07μg/ml、不高于约0.06μg/ml、不高于约0.05μg/ml、不高于约0.04μg/ml、不高于约0.03μg/ml、不高于约0.02μg/ml或不高于约0.01μg/ml或以下)与表达于CHOK1细胞表面的人Siglec15特异性结合。 In the present application, the isolated antigen-binding protein may have an EC 50 value of about 0.2 μg/ml or less in flow cytometry (for example, the EC 50 value is not higher than about 0.2 μg/ml, not More than about 0.15 μg/ml, not more than about 0.13 μg/ml, not more than about 0.12 μg/ml, not more than about 0.1 μg/ml, not more than about 0.09 μg/ml, not more than about 0.08 μg /ml, not higher than about 0.07 μg/ml, not higher than about 0.06 μg/ml, not higher than about 0.05 μg/ml, not higher than about 0.04 μg/ml, not higher than about 0.03 μg/ml, not higher at about 0.02 μg/ml or not higher than about 0.01 μg/ml or lower) specifically binds to human Siglec15 expressed on the surface of CHOK1 cells.
在本申请中，所述分离的抗原结合蛋白在ELISA检测中，可以以约0.2μg/ml或以下的EC 50值(例如，所述EC 50值为不高于约0.2μg/ml、不高于约0.19μg/ml、不高于约0.18μg/ml、不高于约0.17μg/ml、不高于约0.16μg/ml、不高于约0.15μg/ml、不高于约0.1μg/ml、不高于约0.09μg/ml、不高于约0.08μg/ml、不高于约0.07μg/ml、不高于约0.06μg/ml、不高于约0.05μg/ml或不高于约0.01μg/ml或以下)与人Siglec15特异性结合。 In the present application, the isolated antigen-binding protein may have an EC 50 value of about 0.2 μg/ml or less in ELISA detection (for example, the EC 50 value is not higher than about 0.2 μg/ml, not higher At about 0.19 μg/ml, not higher than about 0.18 μg/ml, not higher than about 0.17 μg/ml, not higher than about 0.16 μg/ml, not higher than about 0.15 μg/ml, not higher than about 0.1 μg/ml ml, not more than about 0.09 μg/ml, not more than about 0.08 μg/ml, not more than about 0.07 μg/ml, not more than about 0.06 μg/ml, not more than about 0.05 μg/ml or not more than About 0.01 μg/ml or less) specifically binds to human Siglec15.
在本申请中，所述分离的抗原结合蛋白在ELISA检测中，可以以约0.1μg/ml或以下的EC 50值(例如，所述EC 50值为不高于约0.1μg/ml、不高于约0.09μg/ml、不高于约0.08μg/ml、不高于约0.07μg/ml、不高于约0.06μg/ml、不高于约0.05μg/ml、不高于约0.04μg/ml、不高于约0.03μg/ml、不高于约0.02μg/ml或不高于约0.01μg/ml或以下)与猴Siglec15特异性结合。 In the present application, the isolated antigen-binding protein may have an EC 50 value of about 0.1 μg/ml or less in ELISA detection (for example, the EC 50 value is not higher than about 0.1 μg/ml, not higher At about 0.09 μg/ml, not higher than about 0.08 μg/ml, not higher than about 0.07 μg/ml, not higher than about 0.06 μg/ml, not higher than about 0.05 μg/ml, not higher than about 0.04 μg/ml ml, not higher than about 0.03 μg/ml, not higher than about 0.02 μg/ml, or not higher than about 0.01 μg/ml or less) specifically binds to monkey Siglec15.
在本申请中，所述分离的抗原结合蛋白在ELISA检测中，可以以约0.1μg/ml或以下的EC 50值(例如，所述EC 50值为不高于约0.1μg/ml、不高于约0.09μg/ml、不高于约0.08μg/ml、不高于约0.07μg/ml、不高于约0.06μg/ml、不高于约0.05μg/ml、不高于约0.04μg/ml、不高于约0.03μg/ml、不高于约0.02μg/ml或不高于约0.01μg/ml或以下)与鼠Siglec15特异性结合。 In the present application, the isolated antigen-binding protein may have an EC 50 value of about 0.1 μg/ml or less in ELISA detection (for example, the EC 50 value is not higher than about 0.1 μg/ml, not higher At about 0.09 μg/ml, not higher than about 0.08 μg/ml, not higher than about 0.07 μg/ml, not higher than about 0.06 μg/ml, not higher than about 0.05 μg/ml, not higher than about 0.04 μg/ml ml, not higher than about 0.03 μg/ml, not higher than about 0.02 μg/ml, or not higher than about 0.01 μg/ml or less) specifically binds to murine Siglec15.
在本申请中，所述分离的抗原结合蛋白可以解除Siglec15对血液中PBMC的增殖抑制。例如，所述解除Siglec15对血液中PBMC的增殖抑制可以包括解除Siglec15对T细胞的增殖 抑制。例如，所述对解除Siglec15对T细胞的增殖抑制可以包括促进CD8 +和CD4 +T细胞的增殖和/或促进IFN-γ的表达。 In the present application, the isolated antigen-binding protein can relieve Siglec15 from inhibiting the proliferation of PBMC in blood. For example, releasing the inhibition of Siglec15 on the proliferation of PBMCs in blood may include releasing the inhibition of Siglec15 on the proliferation of T cells. For example, the release of Siglec15's inhibition of T cell proliferation may include promoting the proliferation of CD8 + and CD4 + T cells and/or promoting the expression of IFN-γ.
在本申请中，所述分离的抗原结合蛋白抑制肿瘤细胞的生长和/或增殖。例如，所述抑制肿瘤细胞的生长和/或增殖包括在肿瘤微环境中，所述分离的抗原结合蛋白通过靶向免疫细胞表面的所述Siglec15，解除所述Siglec15对免疫细胞的抑制，从而激活免疫细胞对肿瘤细胞生长和/或增殖的抑制。In the present application, the isolated antigen binding protein inhibits the growth and/or proliferation of tumor cells. For example, the inhibition of the growth and/or proliferation of tumor cells includes that in the tumor microenvironment, the isolated antigen-binding protein releases the inhibition of Siglec15 on immune cells by targeting the Siglec15 on the surface of immune cells, thereby activating Inhibition of tumor cell growth and/or proliferation by immune cells.
一方面，本申请提供一种分离的抗原结合蛋白。所述的分离的抗原结合蛋白可以包括抗体或抗原结合片段。例如，在本申请中，所述的抗原结合片段可以包括：Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv，VHH和/或dAb。例如，在本申请中，所述的抗体可以包括：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和/或全人源抗体。 In one aspect, the application provides an isolated antigen binding protein. The isolated antigen binding protein may comprise an antibody or an antigen binding fragment. For example, in this application, the antigen-binding fragments may include: Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb. For example, in this application, the antibodies may include: monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
在本申请中，所述的分离的抗原结合蛋白可以为单域抗体。In this application, the isolated antigen-binding protein may be a single domain antibody.
一方面，本申请提供一种分离的抗原结合蛋白，其可以包含抗体重链可变区VH中的至少一个CDR，所述VH可以包含SEQ ID NO:34所示的氨基酸序列。In one aspect, the application provides an isolated antigen-binding protein, which may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:34.
例如，所述VH可以包含SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:28中任一项所示的氨基酸序列。在本申请中，所述分离的抗原结合蛋白的HCDR可以通过任何形式划分，只要VH与SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:28中任一项所示的氨基酸序列相同，以任何形式划分得到的HCDR都可落入本申请的保护范围内。For example, the VH can comprise SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: The amino acid sequence shown in any one of ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:28. In this application, the HCDR of the isolated antigen-binding protein can be divided by any form, as long as VH is compatible with SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO :17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:28 shown in any one The amino acid sequences of the HCDRs are the same, and the HCDRs obtained by dividing them in any form can fall within the protection scope of the present application.
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码***来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码***为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码***确定出CDR区。使用不同的编码***，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或***；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约 96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述分离的抗原结合蛋白通过IMGT编码***定义。The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region may make contacts with the antigen or antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems. In this application, the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids . For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology. In certain embodiments, the isolated antigen binding proteins described herein are defined by the IMGT coding system.
在本申请中，所述抗原结合蛋白可以包含重链可变区VH，所述VH可以包含HCDR1、HCDR2和HCDR3中的至少一个、两个或三个。In the present application, the antigen-binding protein may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR3，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR3序列可根据IMGT编码***定义。In the present application, the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1. For example, the HCDR3 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR2，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR2序列可根据IMGT编码***定义。In the present application, the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2. For example, the HCDR2 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1，所述HCDR1可以包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1可以为G或R，X 2可以为F或S，X 3可以为I或T，X 4可以为F，X 5可以为S。例如，所述分离的抗原结合蛋白的HCDR1序列可根据IMGT编码***定义。 In the present application, the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein , X1 can be G or R, X2 can be F or S, X3 can be I or T, X4 can be F, X5 can be S. For example, the HCDR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
例如，所述抗原结合蛋白的HCDR1与SEQ ID NO:30所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1,X 2,X 3,X 4,X 5。 For example, compared with the sequence shown in SEQ ID NO:30, HCDR1 of the antigen binding protein has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 ,X 3 ,X 4 ,X 5 .
[X 1][X 2][X 3][X 4][X 5]NYA(SEQ ID NO:30)，其中，X 1可以是G或R，X 2可以是F或S，X 3可以是I或T，X 4可以是F，X 5可以是S。 [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA (SEQ ID NO:30), wherein, X 1 can be G or R, X 2 can be F or S, and X 3 can be Is I or T, X 4 can be F, X 5 can be S.
例如，所述HCDR1可以包含SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:22中任一项所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR1序列可根据IMGT编码***定义。For example, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:22. For example, the HCDR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，其中所述HCDR1可以包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。例如，所述抗原结合蛋白可包括抗体Ab0到Ab11或与其具有相同HCDR(例如，与其具有相同的HCDR1-3)的抗原结合片段。 In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA Amino acid sequence, wherein, X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, X5 is S, and the HCDR2 may comprise the amino acid shown in SEQ ID NO:2 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22. For example, the antigen binding protein may comprise antibodies Ab0 to Ab11 or an antigen binding fragment having the same HCDRs (eg, having the same HCDR1-3) therewith.
例如，所述抗原结合蛋白的VH可以包含框架区H-FR1，H-FR2，H-FR3和/或H-FR4。For example, the VH of the antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3 and/or H-FR4.
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C末端与所述 HCDR1的N末端直接或间接相连，且所述H-FR1可以包含SEQ ID NO:29所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR1序列可根据IMGT编码***定义。In the present application, the isolated antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 may comprise SEQ ID NO : the amino acid sequence shown in 29. For example, the H-FR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
例如，所述抗原结合蛋白的H-FR1与SEQ ID NO:29所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1。 For example, compared with the sequence shown in SEQ ID NO: 29, H-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 .
[X 1]VQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:29)，其中，X 1可以是A或E。 [X 1 ] VQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:29), wherein X 1 can be A or E.
例如，所述抗原结合蛋白的H-FR1可以包含SEQ ID NO:9或SEQ ID NO:9所示的氨基酸序列。For example, the H-FR1 of the antigen binding protein may comprise SEQ ID NO:9 or the amino acid sequence shown in SEQ ID NO:9.
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2可以包含SEQ ID NO:32所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR2序列可根据IMGT编码***定义。In the present application, the isolated antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise SEQ ID NO:32 amino acid sequence. For example, the H-FR2 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
例如，所述抗原结合蛋白的H-FR2与SEQ ID NO:32所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1，X 2，X 3，X 4。 For example, compared with the sequence shown in SEQ ID NO:32, H-FR2 of the antigen-binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 , X 3 , X 4 .
MEWYRQAPGK[X 1][X 2]ELVAH(SEQ ID NO:32)，其中，X 1可以是G或Q，X 2可以是L或R。 MEWYRQAPGK[X 1 ][X 2 ]ELVAH (SEQ ID NO:32), wherein X 1 can be G or Q, and X 2 can be L or R.
例如，所述抗原结合蛋白的H-FR2可以包含SEQ ID NO:5或SEQ ID NO:10所示的氨基酸序列。For example, the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10.
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3可以包含SEQ ID NO:31所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR3序列可根据IMGT编码***定义。In the present application, the isolated antigen binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise SEQ ID NO:31 amino acid sequence. For example, the H-FR3 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
例如，所述抗原结合蛋白的H-FR3与SEQ ID NO:31所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1，X 2，X 3，X 4。 For example, compared with the sequence shown in SEQ ID NO: 31, H-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 , X 3 , X 4 .
FY[X 1]DSVKGRFTISRDN[X 2]KNT[X 3]YLQMNSL[X 4][X 5]EDTAVYYC(SEQ ID NO:31)，其中，X 1可以是A或V，X 2可以是A或N或S，X 3可以是L或V，X 4可以是K或R,X 5可以是A或P。 FY[X 1 ]DSVKGRFTISRDN[X 2 ]KNT[X 3 ]YLQMNSL[X 4 ][X 5 ]EDTAVYYC (SEQ ID NO:31), wherein X 1 can be A or V, X 2 can be A or N Or S, X 3 can be L or V, X 4 can be K or R, X 5 can be A or P.
例如，所述抗原结合蛋白的H-FR3可以包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18或SEQ ID NO:27所示的氨基酸序列。For example, the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:18 or SEQ ID NO:27.
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4可以包含SEQ ID NO:33所示的氨基酸序列。例如，所述分离的抗原结合蛋白的H-FR4序列可根据IMGT编码***定义。In the present application, the isolated antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 may comprise SEQ ID NO : the amino acid sequence shown in 33. For example, the H-FR4 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
例如，所述抗原结合蛋白的H-FR4与SEQ ID NO:33所示的序列相比，在选自下组的一 个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1。 For example, compared with the sequence shown in SEQ ID NO: 33, H-FR4 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 .
WGQGT[X 1]VTVSS(SEQ ID NO:33)，其中，X 1可以是M或Q。 WGQGT[X 1 ]VTVSS (SEQ ID NO: 33), wherein X 1 can be M or Q.
例如，所述抗原结合蛋白的H-FR4可以包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。For example, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可以包含SEQ ID NO:29所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:32所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:31所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:33所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 29, wherein The H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:32, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:33 amino acid sequence.
在本申请中，所述的分离的抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1可以包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10或SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18或SEQ ID NO:27所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。In the present application, the isolated antigen-binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise SEQ ID NO:4 or SEQ ID NO:9. The amino acid sequence shown, the H-FR2 can include the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 5, the H-FR3 can include SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID The amino acid sequence shown in NO:14, SEQ ID NO:18 or SEQ ID NO:27, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
在本申请中，所述的分离的抗原结合蛋白可以包含选自任一组H-FR1，H-FR2，H-FR3和H-FR4的抗原结合蛋白：例如，In the present application, the isolated antigen binding protein may comprise an antigen binding protein selected from any group of H-FR1, H-FR2, H-FR3 and H-FR4: for example,
1)所述H-FR1可以包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列；1) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:7;
2)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；2) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
3)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；3) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
4)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；4) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
5)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；5) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
6)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；6) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12;
7)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；或，7) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence shown, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12; or,
8)所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:27所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列。8) The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:27 The amino acid sequence shown, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:12.
例如，所述分离的抗原结合蛋白的重链可变区可以包含抗体重链可变区VH中的至少一个CDR和框架区中的至少一个H-FR。For example, the heavy chain variable region of the isolated antigen binding protein can comprise at least one CDR in the antibody heavy chain variable region VH and at least one H-FR in the framework region.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和/或H-FR4，且所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，所述H-FR2位于所述HCDR1和所述HCDR2之间，所述H-FR3位于所述HCDR2和所述HCDR3之间，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连。In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, and the C-terminus of the H-FR1 is connected to the The N-terminal of the HCDR1 is directly or indirectly connected, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminal of the H-FR4 directly or indirectly connected to the C-terminus of the HCDR3.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和/或H-FR4，所述HCDR1可以包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:29所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:32所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:31所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:33所示的氨基酸序列。 In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, and the HCDR1 may comprise [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]An amino acid sequence shown in NYA, wherein X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, X 5 is S, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:29 The amino acid sequence of the H-FR2 can comprise the amino acid sequence shown in SEQ ID NO:32, the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:31 The amino acid sequence shown in NO:33.
在本申请中，所述的分离的抗原结合蛋白可以包含HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和/或H-FR4，其中所述HCDR1可以包含SEQ ID NO:3、SEQ ID NO:20和/或SEQ ID NO:22所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:4和/或SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10和/或SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18和/或SEQ ID NO:27所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7和/或SEQ ID NO:12所示的氨基酸序列。In the present application, said isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and/or H-FR4, wherein said HCDR1 may comprise SEQ ID NO:3 , SEQ ID NO:20 and/or the amino acid sequence shown in SEQ ID NO:22, the HCDR2 can include the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 can include the amino acid sequence shown in SEQ ID NO:1 Sequence, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4 and/or SEQ ID NO:9, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10 and/or SEQ ID NO:5 The amino acid sequence of the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 18 and/or SEQ ID NO: 27, and the The H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7 and/or SEQ ID NO:12.
在本申请中，所述的分离的抗原结合蛋白可以包含选自任一组HCDR1，HCDR2，HCDR3，H-FR1，H-FR2，H-FR3和H-FR4的抗原结合蛋白：例如，In the present application, the isolated antigen binding protein may comprise an antigen binding protein selected from any group of HCDR1, HCDR2, HCDR3, H-FR1, H-FR2, H-FR3 and H-FR4: for example,
1)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:7所示的氨基酸序列；1) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:4, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7;
2)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；2) the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
3)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；3) the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
4)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；4) the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
5)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；5) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:14 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
6)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所 示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；6) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12;
7)所述HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列；或，7) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 amino acid sequence, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 12; or,
8)所述HCDR1可以包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列。8) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:22, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:11 The amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
9)所述HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列。9) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
10)所述HCDR1可以包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列。10) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:22, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
11)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3可以包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列。11) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
12)所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列，所述H-FR1可以包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:5所示 的氨基酸序列，所述H-FR3可以包含SEQ ID NO:27所示的氨基酸序列，且所述H-FR4可以包含SEQ ID NO:12所示的氨基酸序列。12) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1, The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:9, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:27 The amino acid sequence, and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 12.
在本申请中，所述的分离的抗原结合蛋白可以包含重链可变区VH，所述VH可以包含SEQ ID NO:34所示的氨基酸序列。例如，所述抗原结合蛋白可以包含VH，所述VH与SEQ ID NO:34所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1，X 2，X 3，X 4，X 5，X 6，X 7，X 8，X 9，X 10，X 11，X 12，X 13，X 14。 In the present application, the isolated antigen-binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:34. For example, the antigen-binding protein may comprise a VH having amino acid substitutions at one or more amino acids selected from the group (eg, conservative amino acid substitutions, etc.) compared to the sequence shown in SEQ ID NO: 34 : X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 .
[X 1]VQLVESGGGLVQPGGSLRLSCAAS[X 2][X 3][X 4][X 5][X 6]NYAMEWYRQAPGK[X 7][X 8]ELVAHISRDVTTFY[X 9]DSVKGRFTISRDN[X10]KNT[X11]YLQMNSL[X 12][X 13]EDTAVYYCYFPFFDLWGQGT[X 14]VTVSS(SEQ ID NO:34)，其中，X 1可以是A或E，X 2可以是G或R，X 3可以是F或S，X 4可以是I或T，X 5可以是F，X 6可以是S，X 7可以是G或Q，X 8可以是L或R，X 9可以是A或V，X 10可以是A、N或S，X 11可以是L或V，X 12可以是K或R，X 13可以是A或P，X 14可以是M或Q。 [X 1 ]VQLVESGGGLVQPGGSLRLSCAAS[X 2 ][X 3 ][X 4 ][X 5 ][X 6 ]NYAMEWYRQAPGK[X 7 ][X 8 ]ELVAHISRDVTTFY[X 9 ]DSVKGRFTISRDN[X10]KNT[X11]YLQMNSL[X 12 ][X 13 ]EDTAVYYCYFPFFDLWGQGT[X 14 ]VTVSS (SEQ ID NO:34), wherein, X 1 can be A or E, X 2 can be G or R, X 3 can be F or S, and X 4 can be I or T, X 5 can be F, X 6 can be S, X 7 can be G or Q, X 8 can be L or R, X 9 can be A or V, X 10 can be A, N or S, X 11 can be L or V, X 12 can be K or R, X 13 can be A or P, and X 14 can be M or Q.
例如，所述抗原结合蛋白的重链可变区VH可以包含Ab0(氨基酸序列如SEQ ID NO:8所示)、Ab1(氨基酸序列如SEQ ID NO:13所示)、Ab2(氨基酸序列如SEQ ID NO:15所示)、Ab3(氨基酸序列如SEQ ID NO:16所示)、Ab4(氨基酸序列如SEQ ID NO:17所示)、Ab5(氨基酸序列如SEQ ID NO:19所示)、Ab6(氨基酸序列如SEQ ID NO:21所示)、Ab7(氨基酸序列如SEQ ID NO:23所示)、Ab8(氨基酸序列如SEQ ID NO:24所示)、Ab9(氨基酸序列如SEQ ID NO:25所示)、Ab10(氨基酸序列如SEQ ID NO:26所示)或Ab11(氨基酸序列如SEQ ID NO:28所示)中任一项所示的氨基酸序列。For example, the heavy chain variable region VH of the antigen binding protein may comprise Ab0 (amino acid sequence shown in SEQ ID NO: 8), Ab1 (amino acid sequence shown in SEQ ID NO: 13), Ab2 (amino acid sequence shown in SEQ ID NO: 13), Ab2 (amino acid sequence shown in SEQ ID NO: ID NO:15), Ab3 (amino acid sequence as shown in SEQ ID NO:16), Ab4 (amino acid sequence as shown in SEQ ID NO:17), Ab5 (amino acid sequence as shown in SEQ ID NO:19), Ab6 (amino acid sequence shown in SEQ ID NO: 21), Ab7 (amino acid sequence shown in SEQ ID NO: 23), Ab8 (amino acid sequence shown in SEQ ID NO: 24), Ab9 (amino acid sequence shown in SEQ ID NO 25), the amino acid sequence shown in any one of Ab10 (amino acid sequence as shown in SEQ ID NO: 26) or Ab11 (amino acid sequence as shown in SEQ ID NO: 28).
例如，所述的分离的抗原结合蛋白可包括与其具有相同HCDR(例如，与其具有相同的HCDR1-3)的抗原结合片段。For example, the isolated antigen binding protein can include an antigen binding fragment having the same HCDRs (eg, having the same HCDR1-3) as it.
在本申请中，所述的分离的抗原结合蛋白，其可以包含重链恒定区，且所述重链恒定区可以包含源自IgG、IgM、IgA、IgD和/或IgE的恒定区。例如，所述重链恒定区可以包含源自IgG的恒定区。In the present application, the isolated antigen-binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise a constant region derived from IgG, IgM, IgA, IgD and/or IgE. For example, the heavy chain constant region may comprise an IgG-derived constant region.
在本申请中，所述的分离的抗原结合蛋白，其中所述重链恒定区可以包括源自人IgG的恒定区。例如，所述重链恒定区可以包含SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37和/或SEQ ID NO:38所示的氨基酸序列。In the present application, the isolated antigen-binding protein, wherein the heavy chain constant region may comprise a constant region derived from human IgG. For example, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and/or SEQ ID NO:38.
在本申请中，所述分离的抗原结合蛋白还可与参比抗体竞争结合所述人Siglec15蛋白，所述参比抗体可以包含重链可变区VH，所述VH可以包含HCDR1、HCDR2和/或HCDR3中的至少一个、两个或三个。In the present application, the isolated antigen-binding protein can also compete with a reference antibody for binding to the human Siglec15 protein, and the reference antibody can comprise a heavy chain variable region VH, and the VH can comprise HCDR1, HCDR2 and/or Or at least one, two or three of HCDR3.
在本申请中，所述参比抗体的HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述参比抗体的HCDR3的序列可以根据IMGT编码***定义。In the present application, the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:1. For example, the sequence of the HCDR3 of the reference antibody can be defined according to the IMGT coding system.
在本申请中，所述参比抗体的HCDR2可以包含SEQ ID NO:2所示的氨基酸序列。例如，所述参比抗体的HCDR2的序列可根据IMGT编码***定义。In the present application, the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:2. For example, the sequence of the HCDR2 of the reference antibody can be defined according to the IMGT coding system.
在本申请中，所述参比抗体的HCDR1可以包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S。例如，所述参比抗体的HCDR1的序列可以根据IMGT编码***定义。 In the present application, the HCDR1 of the reference antibody may comprise the amino acid sequence represented by [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, and X 5 is S. For example, the sequence of HCDR1 of the reference antibody can be defined according to the IMGT coding system.
例如，所述参比抗体的HCDR1与SEQ ID NO:30所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1,X 2,X 3,X 4,X 5。 For example, compared with the sequence shown in SEQ ID NO:30, the HCDR1 of the reference antibody has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 2 ,X 3 ,X 4 ,X 5 .
[X 1][X 2][X 3][X 4][X 5]NYA(SEQ ID NO:30)，其中，X 1可以是G或R,X 2可以是F或S,X 3可以是I或T,X 4可以是F,X 5可以是S。 [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA (SEQ ID NO:30), wherein, X 1 can be G or R, X 2 can be F or S, and X 3 can be It is I or T, X 4 can be F, and X 5 can be S.
例如，所述参比抗体的HCDR1可以包含SEQ ID NO:3、SEQ ID NO:20和SEQ ID NO:22中任一项所示的氨基酸序列。例如，所述分离的抗原结合蛋白的HCDR1序列可根据IMGT编码***定义。For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 22. For example, the HCDR1 sequence of the isolated antigen binding protein can be defined according to the IMGT coding system.
例如，所述参比抗体的HCDR1可以包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S；所述HCDR2可以包含SEQ ID NO:2所示的氨基酸序列；且所述HCDR3可以包含SEQ ID NO:1所示的氨基酸序列。例如，所述参比抗体可包括抗体Ab0到Ab11或与其具有相同HCDR(例如，与其具有相同HCDR1-3)的抗原结合蛋白。 For example, the HCDR1 of the reference antibody may comprise the amino acid sequence shown as [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA, wherein X 1 is G or R, and X 2 is F or S, X3 is I or T, X4 is F, and X5 is S; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:1 amino acid sequence. For example, the reference antibody may comprise antibodies Ab0 to Ab11 or an antigen binding protein having the same HCDRs therewith (eg, having the same HCDR1-3 as them).
在本申请中，所述参比抗体可以包含重链可变区，所述重链可变区可以包含SEQ ID NO:34所示的氨基酸序列。例如，所述参比抗体可以包含VH，所述VH与SEQ ID NO:34所示的序列相比，在选自下组的一个或多个氨基酸处存在氨基酸取代(例如，保守氨基酸取代等)：X 1，X 2，X 3，X 4，X 5，X 6，X 7，X 8，X 9，X 10，X 11，X 12，X 13，X 14。 In the present application, the reference antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:34. For example, the reference antibody may comprise a VH having amino acid substitutions at one or more amino acids selected from the group (eg, conservative amino acid substitutions, etc.) compared to the sequence shown in SEQ ID NO: 34 : X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 .
[X 1]VQLVESGGGLVQPGGSLRLSCAAS[X 2][X 3][X 4][X 5][X 6]NYAMEWYRQAPGK[X 7][X 8]ELVAHISRDVTTFY[X 9]DSVKGRFTISRDN[X10]KNT[X11]YLQMNSL[X 12][X 13]EDTAVYYCYFPFFDLWGQGT[X 14]VTVSS(SEQ ID NO:34)，其中，X 1可以是A或E，X 2可以是G或R，X 3可以是F或S，X 4可以是I或T，X 5可以是F，X 6可以是S，X 7可以是G或Q，X 8可以是L或R，X 9可以是A或V，X 10可以是A、N或S，X 11可以是L或V，X 12可以是K或R，X 13可以是A或P，X 14可以是M或Q。 [X 1 ]VQLVESGGGLVQPGGSLRLSCAAS[X 2 ][X 3 ][X 4 ][X 5 ][X 6 ]NYAMEWYRQAPGK[X 7 ][X 8 ]ELVAHISRDVTTFY[X 9 ]DSVKGRFTISRDN[X10]KNT[X11]YLQMNSL[X 12 ][X 13 ]EDTAVYYCYFPFFDLWGQGT[X 14 ]VTVSS (SEQ ID NO:34), wherein, X 1 can be A or E, X 2 can be G or R, X 3 can be F or S, and X 4 can be I or T, X 5 can be F, X 6 can be S, X 7 can be G or Q, X 8 can be L or R, X 9 can be A or V, X 10 can be A, N or S, X 11 can be L or V, X 12 can be K or R, X 13 can be A or P, and X 14 can be M or Q.
在本申请中，所述参比抗体的重链可变区可以包含Ab0(氨基酸序列如SEQ ID NO:8所 示)、Ab1(氨基酸序列如SEQ ID NO:13所示)、Ab2(氨基酸序列如SEQ ID NO:15所示)、Ab3(氨基酸序列如SEQ ID NO:16所示)、Ab4(氨基酸序列如SEQ ID NO:17所示)、Ab5(氨基酸序列如SEQ ID NO:19所示)、Ab6(氨基酸序列如SEQ ID NO:21所示)、Ab7(氨基酸序列如SEQ ID NO:23所示)、Ab8(氨基酸序列如SEQ ID NO:24所示)、Ab9(氨基酸序列如SEQ ID NO:25所示)、Ab10(氨基酸序列如SEQ ID NO:26所示)和Ab11(氨基酸序列如SEQ ID NO:28所示)中任一项所示的氨基酸序列。In the present application, the heavy chain variable region of the reference antibody may comprise Ab0 (amino acid sequence shown in SEQ ID NO: 8), Ab1 (amino acid sequence shown in SEQ ID NO: 13), Ab2 (amino acid sequence As shown in SEQ ID NO:15), Ab3 (amino acid sequence as shown in SEQ ID NO:16), Ab4 (amino acid sequence as shown in SEQ ID NO:17), Ab5 (amino acid sequence as shown in SEQ ID NO:19 ), Ab6 (amino acid sequence as shown in SEQ ID NO:21), Ab7 (amino acid sequence as shown in SEQ ID NO:23), Ab8 (amino acid sequence as shown in SEQ ID NO:24), Ab9 (amino acid sequence as shown in SEQ ID NO:24) The amino acid sequence shown in any one of Ab11 (shown in SEQ ID NO: 28), Ab10 (amino acid sequence shown in SEQ ID NO: 26) and Ab11 (amino acid sequence shown in ID NO: 25).
在本申请中，所述参比抗体可以包含重链恒定区，所述重链恒定区可以包括源自IgG的恒定区和/或源自IgY的恒定区。In the present application, the reference antibody may comprise a heavy chain constant region, which may comprise an IgG-derived constant region and/or an IgY-derived constant region.
例如，所述参比抗体的重链恒定区可以包含SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38中任一项所示的氨基酸序列。For example, the heavy chain constant region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
多肽和免疫缀合物Peptides and Immunoconjugates
另一方面，本申请提供了一种或多种多肽，其可以包含本申请的分离的抗原结合蛋白及其片段。In another aspect, the application provides one or more polypeptides, which may comprise the isolated antigen binding proteins of the application and fragments thereof.
另一方面，本身请提供了一种或多种免疫缀合物，所述免疫缀合物可以包含本申请所述的分离的抗原结合蛋白或所述的多肽。例如，所述免疫缀合物可以包括与抗原结合蛋白融合或偶联的其他的多肽、治疗剂、探针和/或其他抗体。在某些实施方式中，所述免疫缀合物可以包括药学上可接受的标记物、检测剂和/或治疗物。In another aspect, there is provided one or more immunoconjugates, which may comprise the isolated antigen-binding protein or the polypeptide described herein. For example, the immunoconjugate can include other polypeptides, therapeutic agents, probes and/or other antibodies fused or conjugated to the antigen binding protein. In certain embodiments, the immunoconjugate can include a pharmaceutically acceptable label, detection agent, and/or therapeutic.
核酸、载体、细胞和药物组合物Nucleic acids, vectors, cells and pharmaceutical compositions
另一方面，本申请提供了一种或多种分离的核酸分子，所述一种或多种分离的核酸分子可以编码本申请所述的分离的抗原结合蛋白或者本申请所述的多肽。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码所述抗原结合蛋白中的一部分。例如，所述一种或多种核酸分子的每一个核酸分子可以构成一种或多种核酸序列的排列组合。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码一种或多种本申请所述的抗原结合蛋白或其片段的排列组合。(例如，一种或多种核酸分子中的每一个核酸分子可以编码任意完整的HCDR1-HCDR3、FR1-FR4、IgG1-3重链恒定区或它们的片段的一种或多种的排列组合。)In another aspect, the application provides one or more isolated nucleic acid molecules that encode the isolated antigen binding proteins described herein or the polypeptides described herein. For example, each of the one or more nucleic acid molecules may encode the entirety of the antigen binding protein, or may encode a portion of the antigen binding protein. For example, each of the one or more nucleic acid molecules may constitute a permutation of one or more nucleic acid sequences. For example, each of the one or more nucleic acid molecules may encode one or more permutations and combinations of antigen binding proteins or fragments thereof described herein. (For example, each nucleic acid molecule in one or more nucleic acid molecules can encode any complete HCDR1-HCDR3, FR1-FR4, IgG1-3 heavy chain constant region or one or more permutations and combinations of their fragments. )
例如，当所述核酸分子分别编码所述抗原结合蛋白或其片段时，所述核酸分子编码的产物的组合可以是有功能的本申请所述的分离的抗原结合蛋白(例如，所述分离的抗原结合蛋白可以特异地结合Siglec15)。For example, when the nucleic acid molecules separately encode the antigen binding proteins or fragments thereof, the combination of products encoded by the nucleic acid molecules may be a functional isolated antigen binding protein described herein (e.g., the isolated Antigen binding proteins can specifically bind Siglec15).
例如，本申请所述核酸分子的每一个核酸可以为分离的。例如，所述核酸分子可以通过 以下方法产生或合成：(i)体外扩增，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)克隆重组，(iii)纯化的，例如通过酶切、凝胶电泳分级分离和/或胶纯化试剂盒回收，或者(iv)合成的，例如通过人工化学合成。例如，所述分离的核酸可以是通过重组DNA技术制备的核酸分子。For example, each nucleic acid of the nucleic acid molecules described herein can be isolated. For example, the nucleic acid molecule can be produced or synthesized by (i) amplification in vitro, e.g., by polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii) purified, e.g., by Enzymatic digestion, gel electrophoresis fractionation and/or gel purification kit recovery, or (iv) synthetic, such as by artificial chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
另一方面，本申请提供了一种或多种分离的载体，所述一种或多种分离的载体可以包括本申请所述的一种或多种核酸分子和/或其他基因。例如，所述一种或多种分离的载体可以包括噬菌体载体、病毒载体和/或细菌载体。例如，所述一种或多种分离的载体可以是共价闭合环状双链的。例如，所述一种或多种分离的载体可以进入宿主细胞并且自主复制。例如，所述其他基因可以包括允许存在于宿主细胞中和/或适当条件下选择的适当的标记基因和/或控制元件。例如，所述控制元件可以包括：复制起始位点、核糖体结合位点、启动子、增强子、沉默子、多克隆位点和/或终止信号。在某些实施方式中，所述启动子可以包括：用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子和/或CMV启动子。例如，所述标记基因可以包括：抗生素抗性基因、荧光酶标记基因和/或β-半乳糖苷酶基因。In another aspect, the application provides one or more isolated vectors, which may include one or more nucleic acid molecules and/or other genes described herein. For example, the one or more isolated vectors can include phage vectors, viral vectors and/or bacterial vectors. For example, the one or more isolated vectors may be covalently closed circular double-stranded. For example, the one or more isolated vectors can enter a host cell and replicate autonomously. For example, said other genes may include appropriate marker genes and/or control elements allowing their presence in the host cell and/or selection under appropriate conditions. For example, the control elements may include: an origin of replication, a ribosome binding site, a promoter, an enhancer, a silencer, a multiple cloning site, and/or a termination signal. In certain embodiments, the promoters may include: promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, and/or CMV promoters. For example, the marker gene may include: an antibiotic resistance gene, a luciferase marker gene and/or a β-galactosidase gene.
另一方面，本申请提供了一种或多种细胞。例如，所述细胞可以包含本申请所述的分离的抗原结合蛋白和/或其片段、所述的多肽、所述的免疫缀合物、所述的一种或多种核酸分子和/或所述的一种或多种载体。例如，所述的一种或多种细胞中的每个或每种细胞可以包含一个、一种、多个和/或多种本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中。例如，所述细胞可以包括：原核细胞(例如，大肠杆菌细胞)和/或真核细胞(例如，CHO细胞、HEK293T细胞、Expi293细胞和/或HEK293A细胞。在某些实施方式中，可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，所述方法可包括：热击转化法、瞬时转染法和/或稳定转染法。例如，所述转染法可包括：电穿孔法、脂质体转染法和/或磷酸钙转染法等。在某些实施方式中，所述细胞可以包括通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中使之表达(例如，瞬时表达和/或稳定表达)本申请所述抗原结合蛋白或其片段和/或其他蛋白。例如，在某些实施方式中，所述细胞(例如，Expi293细胞)可以瞬时表达本申请所述抗原结合蛋白。例如，在某些实施方式中，所述细胞(例如，CHOK1细胞)可以稳定表达人Siglec蛋白。例如，所述细胞可以包括CHOK1细胞。例如，所述细胞可以包括感受态的大肠杆菌细胞。例如，所述细胞可以包括哺乳动物细胞。例如，所述细胞可以包括免疫细胞。例如，所述免疫细胞可以包括：外周血单个核细胞(PBMC细胞)、T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞和/或白细胞。In another aspect, the application provides one or more cells. For example, the cells may comprise the isolated antigen binding proteins and/or fragments thereof, the polypeptides, the immunoconjugates, the one or more nucleic acid molecules and/or the described nucleic acid molecules described herein. One or more of the vectors described above. For example, each or each of the one or more cells may comprise one, one, a plurality and/or more of the nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into the host cell. For example, the cells may include: prokaryotic cells (e.g., E. coli cells) and/or eukaryotic cells (e.g., CHO cells, HEK293T cells, Expi293 cells, and/or HEK293A cells. In some embodiments, the Methods known in the art introduce the carrier described in the application into the host cell, and the method may include: heat shock transformation, transient transfection and/or stable transfection. For example, the transfection may Including: electroporation, liposome transfection and/or calcium phosphate transfection, etc. In some embodiments, the cells may include introducing the vectors described in the present application into the cells by methods known in the art. Express (for example, transiently express and/or stably express) the antigen binding protein or fragment thereof and/or other protein described in the application in said host cell.For example, in some embodiments, said cell (for example, Expi293 Cells) can transiently express the antigen-binding protein described in the present application. For example, in certain embodiments, the cells (for example, CHOK1 cells) can stably express human Siglec protein. For example, the cells can include CHOK1 cells. For example, Described cell can comprise the escherichia coli cell of competence. For example, described cell can comprise mammalian cell. For example, described cell can comprise immune cell. For example, described immune cell can comprise: Peripheral blood mononuclear cell (PBMC cell ), T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes and/or leukocytes.
另一方面，本申请提供了一种药物组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、所述的分离的核酸分子、所述的载体、所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。在本申请中，所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂。除非与本申请所述的细胞不相容，否则任何常规介质或试剂均可以考虑用于本申请的药物组合物中。在本申请中，所述药学上可接受的赋形剂可以包括在药物制剂中除主药以外的附加物，也可称为辅料。例如，所述赋形剂可以包括片剂中的粘合剂、填充剂、崩解剂、润滑剂。例如，所述赋形剂可以包括中药丸剂中的酒、醋、药汁等。例如，所述赋形剂可以包括半固体制剂软膏剂、霜剂中的基质部分。例如，所述赋形剂可以包括液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂。In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or pharmaceutically acceptable adjuvants and / or excipients. In this application, the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional media or reagents are contemplated for use in the pharmaceutical compositions of the present application. In this application, the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, and may also be referred to as auxiliary materials. For example, the excipients may include binders, fillers, disintegrants, lubricants in tablets. For example, the excipients may include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills. For example, the excipient may comprise the base part of a semi-solid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.
药物组合drug combination
另一方面，本申请提供了一种药物组合，其包含本申请所述分离的抗原结合蛋白和免疫检查点抑制剂。In another aspect, the present application provides a pharmaceutical combination comprising the isolated antigen-binding protein described in the present application and an immune checkpoint inhibitor.
在本申请中，所述免疫检测点抑制剂可包括抑制PD-1/PD-L1相互作用的物质。例如，所述免疫检测点抑制剂可选择下组：PD-1/PD-L1阻断剂、PD-1拮抗剂、PD-L1拮抗剂、PD-1抑制剂和PD-L1抑制剂。In this application, the immune checkpoint inhibitor may include substances that inhibit the interaction of PD-1/PD-L1. For example, the immune checkpoint inhibitor can be selected from the group consisting of PD-1/PD-L1 blockers, PD-1 antagonists, PD-L1 antagonists, PD-1 inhibitors and PD-L1 inhibitors.
例如，所述PD-1/PD-L1阻断剂可选自下组：BMS202(PD-1/PD-L1抑制剂2)、BMS-1(PD-1/PD-L1抑制剂1)、PD-1/PD-L1抑制剂3、BMS-1166和BMS-1001。For example, the PD-1/PD-L1 blocking agent can be selected from the group consisting of BMS202 (PD-1/PD-L1 inhibitor 2), BMS-1 (PD-1/PD-L1 inhibitor 1), PD-1/PD-L1 Inhibitor 3, BMS-1166 and BMS-1001.
例如，所述PD-1抑制剂可包括抗PD-1抗体。例如，所述PD-L1抑制剂可包括抗PD-L1抗体。For example, the PD-1 inhibitor can include an anti-PD-1 antibody. For example, the PD-L1 inhibitor can include an anti-PD-L1 antibody.
例如，所述抗PD-1抗体可选自下组：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。例如，所述抗PD-L1抗体可选自下组：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。For example, the anti-PD-1 antibody can be selected from the group consisting of Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimumab) ), Sintilimab (Sintilimab) and Tislelizumab (Tislelizumab). For example, the anti-PD-L1 antibody may be selected from the group consisting of Durvalumab (Dervalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的HCDR3：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise HCDR3 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的HCDR2：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普 利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise HCDR2 of an antibody selected from the following group: Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的HCDR1：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise HCDR1 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的LCDR3：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise LCDR3 of an antibody selected from the following group: Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的LCDR2：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise LCDR2 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的LCDR1：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise LCDR1 of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的VH：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise the VH of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Karelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-1抗体可包含选自下组的抗体的VL：Nivolumab(纳武单抗)、Pembrolizumab(帕博利珠单抗)、Camrelizumab(卡瑞利珠单抗)、Toripalimab(特瑞普利单抗)、Sintilimab(信迪利单抗)和Tislelizumab(替雷利珠单抗)。In the present application, the anti-PD-1 antibody may comprise the VL of an antibody selected from the following group: Nivolumab (Navolumab), Pembrolizumab (Pembrolizumab), Camrelizumab (Carrelizumab), Toripalimab (Tripalimab), Sintilimab (Sintilimab), and Tislelizumab (Tislelizumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的HCDR3：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise HCDR3 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的HCDR2：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise HCDR2 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的HCDR1：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise HCDR1 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的LCDR3：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise LCDR3 of an antibody selected from the group consisting of Durvalumab (Dirvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的LCDR2：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise LCDR2 of an antibody selected from the group consisting of Durvalumab (Dirvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的LCDR1：Durvalumab(德瓦鲁 单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise LCDR1 of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的VH：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise the VH of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-L1抗体可包含选自下组的抗体的VL：Durvalumab(德瓦鲁单抗)、Atezolizumab(阿替利珠单抗)和avelumab(阿维单抗)。In the present application, the anti-PD-L1 antibody may comprise the VL of an antibody selected from the group consisting of Durvalumab (Durvalumab), Atezolizumab (Atezolizumab) and avelumab (Avelumab).
在本申请中，所述抗PD-1抗体可包含HCDR3，所述HCDR3可包含SEQ ID NO:39所示的氨基酸序列。In the present application, the anti-PD-1 antibody may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:39.
在本申请中，所述抗PD-1抗体包含HCDR2，所述HCDR2包含SEQ ID NO:40所示的氨基酸序列。In the present application, the anti-PD-1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:40.
在本申请中，所述抗PD-1抗体包含HCDR1，所述HCDR1包含SEQ ID NO:41所示的氨基酸序列。In the present application, the anti-PD-1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41.
在本申请中，所述抗PD-1抗体包含重链可变区VH，所述VH包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:39所示的氨基酸序列；所述HCDR2包含SEQ ID NO:40所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:41所示的氨基酸序列。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗体。In the present application, the anti-PD-1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 39; the HCDR2 comprises SEQ ID NO: 39 The amino acid sequence shown in ID NO:40; And described HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41. For example, the anti-PD-1 antibody can comprise pembrolizumab or an antibody that has the same HCDR3 as it (eg, has the same HCDR1-3 as it).
在本申请中，所述抗PD-1抗体可包含重链可变区VH，所述VH可包含SEQ ID NO:42所示的氨基酸序列。In the present application, the anti-PD-1 antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:42.
在本申请中，所述抗PD-1抗体可包含LCDR3，所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。In the present application, the anti-PD-1 antibody may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43.
在本申请中，所述抗PD-1抗体可包含LCDR2，所述LCDR2可包含SEQ ID NO:44所示的氨基酸序列。In the present application, the anti-PD-1 antibody may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:44.
在本申请中，所述抗PD-1抗体可包含LCDR1，所述LCDR1可包含SEQ ID NO:45所示的氨基酸序列。In the present application, the anti-PD-1 antibody may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:45.
在本申请中，所述抗PD-1抗体可包含轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3，所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:44所示的氨基酸序列；以及所述LCDR1可包含SEQ ID NO:45所示的氨基酸序列。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗体。In the present application, the anti-PD-1 antibody may comprise a light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 43; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:44; and said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:45. For example, the anti-PD-1 antibody can comprise pembrolizumab or an antibody that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
在本申请中，所述抗PD-1抗体可包含轻链可变区VL，所述VL可包含SEQ ID NO:46 所示的氨基酸序列。In the present application, the anti-PD-1 antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:46.
在本申请中，所述抗PD-1抗体可包含重链和轻链，所述重链可包含HCDR1-3以及H-FR1-4，且所述轻链可包含LCDR1-3以及L-FR1-4。例如，所述HCDR1可包含SEQ ID NO:41所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:40所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:39所示的氨基酸序列；所述LCDR1可包括SEQ ID NO:45所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:44所示的氨基酸序列；所述LCDR3可包含SEQ ID NO:43所示的氨基酸序列。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)和LCDR3(例如，与其具有相同LCDR1-3)的抗原结合蛋白。In the present application, the anti-PD-1 antibody may comprise a heavy chain and a light chain, the heavy chain may comprise HCDR1-3 and H-FR1-4, and the light chain may comprise LCDR1-3 and L-FR1 -4. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:41; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:40; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:39; The LCDR1 can include the amino acid sequence shown in SEQ ID NO:45; the LCDR2 can include the amino acid sequence shown in SEQ ID NO:44; the LCDR3 can include the amino acid sequence shown in SEQ ID NO:43. For example, the anti-PD-1 antibody can comprise pembrolizumab or an antigen binding protein that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as it.
例如，所述抗PD-1抗体的重链可变区可包含SEQ ID NO:42所示的氨基酸序列。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同重链可变区的抗原结合蛋白。例如，所述抗PD-1抗体的轻链可变区可包含SEQ ID NO:46所示的氨基酸序列。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同轻链可变区的抗原结合蛋白。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同重链的抗原结合蛋白。例如，所述抗PD-1抗体可包括帕博利珠单抗或与其具有相同轻链的抗原结合蛋白。For example, the heavy chain variable region of the anti-PD-1 antibody may comprise the amino acid sequence shown in SEQ ID NO:42. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the anti-PD-1 antibody may comprise the amino acid sequence shown in SEQ ID NO:46. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same light chain variable region as it. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same heavy chain as it. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same light chain as it.
在本申请中，所述抗PD-L1抗体可包含HCDR3，所述HCDR3可包含SEQ ID NO:47所示的氨基酸序列。In the present application, the anti-PD-L1 antibody may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:47.
在本申请中，所述抗PD-L1抗体包含HCDR2，所述HCDR2包含SEQ ID NO:48所示的氨基酸序列。In the present application, the anti-PD-L1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:48.
在本申请中，所述抗PD-L1抗体包含HCDR1，所述HCDR1包含SEQ ID NO:49所示的氨基酸序列。In the present application, the anti-PD-L1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49.
在本申请中，所述抗PD-L1抗体包含重链可变区VH，所述VH包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:47所示的氨基酸序列；所述HCDR2包含SEQ ID NO:48所示的氨基酸序列；以及所述HCDR1包含SEQ ID NO:49所示的氨基酸序列。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗体。In the present application, the anti-PD-L1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 47; the HCDR2 comprises SEQ ID NO: 47 The amino acid sequence shown in ID NO:48; And described HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49. For example, the anti-PD-L1 antibody can comprise atezolizumab or an antibody that has the same HCDR3 as it (eg, has the same HCDR1-3 as it).
在本申请中，所述抗PD-L1抗体可包含重链可变区VH，所述VH可包含SEQ ID NO:50所示的氨基酸序列。In the present application, the anti-PD-L1 antibody may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:50.
在本申请中，所述抗PD-L1抗体可包含LCDR3，所述LCDR3可包含SEQ ID NO:51所示的氨基酸序列。In the present application, the anti-PD-L1 antibody may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:51.
在本申请中，所述抗PD-L1抗体可包含LCDR2，所述LCDR2可包含SEQ ID NO:52所示的氨基酸序列。In the present application, the anti-PD-L1 antibody may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:52.
在本申请中，所述抗PD-L1抗体可包含LCDR1，所述LCDR1可包含SEQ ID NO:53所示的氨基酸序列。In the present application, the anti-PD-L1 antibody may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:53.
在本申请中，所述抗PD-L1抗体可包含轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3，所述LCDR3可包含SEQ ID NO:51所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:52所示的氨基酸序列；以及所述LCDR1可包含SEQ ID NO:53所示的氨基酸序列。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同LCDR3(例如，与其具有相同LCDR1-3)的抗体。In the present application, the anti-PD-L1 antibody may comprise a light chain variable region VL, the VL may comprise LCDR1, LCDR2 and LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 51; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:52; and said LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:53. For example, the anti-PD-L1 antibody can comprise atezolizumab or an antibody that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
在本申请中，所述抗PD-L1抗体可包含轻链可变区VL，所述VL可包含SEQ ID NO:54所示的氨基酸序列。In the present application, the anti-PD-L1 antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.
在本申请中，所述抗PD-L1抗体可包含重链和轻链，所述重链可包含HCDR1-3以及H-FR1-4，且所述轻链可包含LCDR1-3以及L-FR1-4。例如，所述HCDR1可包含SEQ ID NO:49所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:48所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:47所示的氨基酸序列；所述LCDR1可包括SEQ ID NO:53所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述LCDR3可包含SEQ ID NO:51所示的氨基酸序列。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)和LCDR3(例如，与其具有相同LCDR1-3)的抗原结合蛋白。In the present application, the anti-PD-L1 antibody may comprise a heavy chain and a light chain, the heavy chain may comprise HCDR1-3 and H-FR1-4, and the light chain may comprise LCDR1-3 and L-FR1 -4. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:49; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:48; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:47; The LCDR1 can include the amino acid sequence shown in SEQ ID NO:53; the LCDR2 can include the amino acid sequence shown in SEQ ID NO:52; the LCDR3 can include the amino acid sequence shown in SEQ ID NO:51. For example, the anti-PD-L1 antibody can comprise atezolizumab or an antigen binding protein that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as atezolizumab.
例如，所述抗PD-L1抗体的重链可变区可包含SEQ ID NO:50所示的氨基酸序列。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同重链可变区的抗原结合蛋白。例如，所述抗PD-L1抗体的轻链可变区可包含SEQ ID NO:54所示的氨基酸序列。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同轻链可变区的抗原结合蛋白。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同重链的抗原结合蛋白。例如，所述抗PD-L1抗体可包括阿替利珠单抗或与其具有相同轻链的抗原结合蛋白。For example, the heavy chain variable region of the anti-PD-L1 antibody may comprise the amino acid sequence shown in SEQ ID NO:50. For example, the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same heavy chain variable region as it. For example, the light chain variable region of the anti-PD-L1 antibody may comprise the amino acid sequence shown in SEQ ID NO:54. For example, the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same light chain variable region as it. For example, the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same heavy chain as it. For example, the anti-PD-L1 antibody may comprise atezolizumab or an antigen binding protein having the same light chain as it.
本申请所述的药物组合能够对肿瘤有效杀伤。The drug combination described in this application can effectively kill tumors.
在本申请中，所述药物组合中的各活性成分可以联合施用。例如，本申请所述的分离的抗原结合蛋白可以与所述免疫检查点抑制剂联合施用。所述联合施用可以是同时施用，也可以是先后施用。所述联合施用可以是各活性成分各自独立地施用一次，也可以是各活性成分各自独立地施用多次。In the present application, each active ingredient in the pharmaceutical combination can be administered in combination. For example, an isolated antigen binding protein described herein can be administered in combination with the immune checkpoint inhibitor. The combined administration can be administered simultaneously or sequentially. The combined administration may be that each active ingredient is independently administered once, or each active ingredient is independently administered multiple times.
试剂盒、方法、用途Kits, methods, uses
另一方面，本申请提供了一种用于检测或测定Siglec15的方法。所述方法可以包括使用本申请所述的分离的抗原结合蛋白。例如，所述方法可以包括：体外方法、离体方法、非诊断或非治疗目的的方法。例如，所述方法可以包括用于非诊断或非治疗目的的检测Siglec蛋白的存在和/或含量的方法，其可包括下述步骤：In another aspect, the present application provides a method for detecting or measuring Siglec15. The method may comprise the use of an isolated antigen binding protein described herein. For example, the methods may include: in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes. For example, the method may include a method for detecting the presence and/or amount of Siglec protein for non-diagnostic or non-therapeutic purposes, which may include the steps of:
1)使样品与本申请的抗原结合蛋白接触；以及1) contacting a sample with an antigen binding protein of the present application; and
2)检测样品结合的所述抗原结合蛋白的存在和/或含量来确定获自受试者的样品中Siglec蛋白的存在和/或表达水平。2) Detecting the presence and/or amount of the antigen-binding protein bound by the sample to determine the presence and/or expression level of the Siglec protein in the sample obtained from the subject.
另一方面，本申请提供了一种检测Siglec蛋白的检测试剂盒。例如，所述检测试剂盒可以包括本申请所述的分离的抗原结合蛋白。在本申请中，所述检测试剂盒可以包括使用说明、一个或多个容器、化学试剂和/或生物大分子。On the other hand, the present application provides a detection kit for detecting Siglec protein. For example, the detection kit can include an isolated antigen binding protein described herein. In the present application, the detection kit may include instructions for use, one or more containers, chemical reagents and/or biomacromolecules.
另一方面，本申请提供了一种所述的分离的抗原结合蛋白在制备试剂盒中的用途，所述试剂盒可用于检测Siglec蛋白的存在和/或含量的方法。在本申请中，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。In another aspect, the present application provides a use of the isolated antigen-binding protein in preparing a kit, and the kit can be used in a method for detecting the presence and/or content of Siglec protein. In this application, the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
另一方面，本申请提供了一种调节免疫应答的方法。所述方法可以包括向有需要的受试者施用有效量的所述的分离的抗原结合蛋白、所述的免疫缀合物、所述的分离的核酸分子、所述的载体、所述的细胞、所述的药物组合物、所述的药物组合、药学上可接受的治疗剂和/或药学上可接受的佐剂和/或赋形剂。在本申请中，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。In another aspect, the present application provides a method of modulating an immune response. The method may comprise administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell , the pharmaceutical composition, the pharmaceutical combination, a pharmaceutically acceptable therapeutic agent and/or a pharmaceutically acceptable adjuvant and/or excipient. In this application, the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
另一方面，本身请提供了一种所述的分离的抗原结合蛋白、所述的免疫缀合物、所述的分离的核酸分子、所述的载体、所述的细胞，所述的药物组合物和/或所述的药物组合在制备预防和/或治疗疾病或病症的药物中的用途。In another aspect, please provide a kind of said isolated antigen binding protein, said immunoconjugate, said isolated nucleic acid molecule, said carrier, said cell, said pharmaceutical combination The application of the substance and/or the drug combination in the preparation of a drug for preventing and/or treating a disease or a disease.
在本申请中，所述疾病或病症可以包括：肿瘤。例如，所述肿瘤可以包括与Siglec15的表达相关的肿瘤。术语“与Siglec15的表达相关的肿瘤”通常是指在肿瘤微环境中Siglec15蛋白的表达上调导致逃避免疫***而形成的肿瘤。所述与Siglec15的表达相关的肿瘤可以是Siglec15阳性的肿瘤。在Siglec15阳性的肿瘤中，与正常细胞相比，肿瘤微环境中的免疫***细胞表面的Siglec15蛋白表达量高约1％、10％、20％、30％、40％、50％、60％、70％、80％或更高。例如，所述肿瘤可以包括实体瘤。In the present application, the diseases or conditions may include: tumors. For example, the tumor can include a tumor associated with expression of Siglec15. The term "tumor associated with the expression of Siglec15" generally refers to the formation of tumors in which the expression of Siglec15 protein is up-regulated in the tumor microenvironment to escape from the immune system. The tumors associated with the expression of Siglec15 may be Siglec15 positive tumors. In Siglec15-positive tumors, compared with normal cells, the expression of Siglec15 protein on the surface of immune system cells in the tumor microenvironment is about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher. For example, the tumor can comprise a solid tumor.
另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白、所述的免疫缀合物、所述的分离的核 酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的药物组合。On the other hand, the present application provides a method for preventing and/or treating a disease or disorder, the method may comprise administering the isolated antigen-binding protein, the immunoconjugate described in the present application to a subject in need compound, the isolated nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination.
在本申请中，所述疾病或病症可以包括：肿瘤。例如，所述肿瘤可以包括与Siglec15的表达相关的肿瘤。例如，所述肿瘤可以包括与Siglec15的表达相关的肿瘤。术语“与Siglec15的表达相关的肿瘤”通常是指在肿瘤微环境中Siglec15蛋白的表达上调导致逃避免疫***而形成的肿瘤。所述与Siglec15的表达相关的肿瘤可以是Siglec15阳性的肿瘤。在Siglec15阳性的肿瘤中，与正常细胞相比，肿瘤微环境中的免疫***细胞表面的Siglec15蛋白表达量高约1％、10％、20％、30％、40％、50％、60％、70％、80％或更高。例如，所述肿瘤可以包括实体瘤。In the present application, the diseases or conditions may include: tumors. For example, the tumor can include a tumor associated with expression of Siglec15. For example, the tumor can include a tumor associated with expression of Siglec15. The term "tumor associated with the expression of Siglec15" generally refers to the formation of tumors in which the expression of Siglec15 protein is up-regulated in the tumor microenvironment to escape from the immune system. The tumors associated with the expression of Siglec15 may be Siglec15 positive tumors. In Siglec15-positive tumors, compared with normal cells, the expression of Siglec15 protein on the surface of immune system cells in the tumor microenvironment is about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher. For example, the tumor can comprise a solid tumor.
另一方面，本申请提供了所述分离的抗原结合蛋白、所述免疫缀合物、所述分离的核酸分子、所述载体、所述细胞、所述药物组合物和/或所述药物组合，其用于预防和/或治疗疾病或病症。In another aspect, the application provides the isolated antigen binding protein, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination , for the prevention and/or treatment of a disease or condition.
在本申请中，所述疾病或病症可以包括：肿瘤。例如，所述肿瘤可以包括与Siglec15的表达相关的肿瘤。例如，所述肿瘤可以包括与Siglec15的表达相关的肿瘤。术语“与Siglec15的表达相关的肿瘤”通常是指在肿瘤微环境中Siglec15蛋白的表达上调导致逃避免疫***而形成的肿瘤。所述与Siglec15的表达相关的肿瘤可以是Siglec15阳性的肿瘤。在Siglec15阳性的肿瘤中，与正常细胞相比，肿瘤微环境中的免疫***细胞表面的Siglec15蛋白表达量高约1％、10％、20％、30％、40％、50％、60％、70％、80％或更高。例如，所述肿瘤可以包括实体瘤。In the present application, the diseases or conditions may include: tumors. For example, the tumor can include a tumor associated with expression of Siglec15. For example, the tumor can include a tumor associated with expression of Siglec15. The term "tumor associated with the expression of Siglec15" generally refers to the formation of tumors in which the expression of Siglec15 protein is up-regulated in the tumor microenvironment to escape from the immune system. The tumors associated with the expression of Siglec15 may be Siglec15 positive tumors. In Siglec15-positive tumors, compared with normal cells, the expression of Siglec15 protein on the surface of immune system cells in the tumor microenvironment is about 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher. For example, the tumor can comprise a solid tumor.
在本申请中，所述有需要的受试者可以包括人类或其他动物。例如，所述其他动物可以包括：猴、鸡、鹅、猫、狗、小鼠和/或大鼠。此外，其他动物也可以包括任何除人类以外的动物物种，例如，家畜动物、家禽动物、啮齿类动物和/或灵长类动物。所述人类可以是高加索人、非洲人、亚洲人、闪族人和/或其他种族。例如，所述人类可以是老年、成年、青少年、儿童或者婴儿。In this application, the subjects in need may include humans or other animals. For example, the other animals may include: monkeys, chickens, geese, cats, dogs, mice and/or rats. Additionally, other animals may also include any animal species other than humans, eg, livestock animals, poultry animals, rodents, and/or primates. The human can be Caucasian, African, Asian, Semitic and/or other ethnicity. For example, the human can be elderly, adult, adolescent, child or infant.
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。Not intending to be limited by any theory, the following examples are only for explaining various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.
实施例Example
实施例1 免疫羊驼 Embodiment 1 immune alpaca
采用重组人Siglec15蛋白(SG5-H5253，ACRO)免疫一只健康的雌性成年羊驼(Alpaca)。首次免疫将500μg重组人Siglec15蛋白与等体积弗氏完全佐剂乳化后在颈部***附近左右 两侧注射；加强免疫采用500μg重组人Siglec15蛋白与等体积弗氏完全或不完全佐剂乳化后在颈部***附近左右两侧注射，并进行几次加强免疫，末次免疫一周后采血，检测抗血清效价。A healthy female adult alpaca (Alpaca) was immunized with recombinant human Siglec15 protein (SG5-H5253, ACRO). For the first immunization, 500 μg recombinant human Siglec15 protein was emulsified with an equal volume of Freund’s complete adjuvant and injected on the left and right sides near the cervical lymph nodes; for booster immunization, 500 μg recombinant human Siglec15 protein was emulsified with an equal volume of Freund’s complete or incomplete adjuvant and then injected in The left and right sides of the neck lymph nodes were injected, and several booster immunizations were carried out. Blood was collected one week after the last immunization to test the antiserum titer.
实施例2 羊驼免疫库的构建Example 2 Construction of alpaca immune library
免疫结束后，收集实施例1中免疫的羊驼外周血，采用淋巴细胞分离液分离得到实施例1中免疫的羊驼外周血中的淋巴细胞；采用TRIzol TM Reagent提取淋巴细胞总RNA；采用cDNA反转录试剂盒(PrimeScript TM II 1st Strand cDNA Synthesis Kit)反转录得到cDNA，巢式PCR扩增羊驼抗体的VHH基因。采用胶纯化试剂盒回收VHH基因片段后采用限制性内切酶Bgl1酶切消化，随后将其克隆至噬菌粒载体pADL-10b，将构建好的克隆产物转化至E.coli TG1电转化感受态细胞，构建VHH基因文库；采用平板梯度稀释法测定库容量为2.2×10 9pfu，菌落PCR结果显示文库***率为97.9％。从上述基因文库中取10-100倍库容量的活细胞进行接种培养，培养至对数期后采用M13K07噬菌体进行救援，救援培养后，离心收集噬菌体，采用PEG-NaCl纯化噬菌体，即得噬菌体展示文库，可直接用于后续筛选。 After the immunization, the peripheral blood of the alpaca immunized in Example 1 was collected, and the lymphocytes in the peripheral blood of the alpaca immunized in Example 1 were obtained by using a lymphocyte separation medium; the total RNA of the lymphocytes was extracted by using TRIzol ™ Reagent; The reverse transcription kit (PrimeScript TM II 1st Strand cDNA Synthesis Kit) was reverse transcribed to obtain cDNA, and the VHH gene of the alpaca antibody was amplified by nested PCR. The VHH gene fragment was recovered with a gel purification kit, digested with restriction endonuclease Bgl1, and then cloned into the phagemid vector pADL-10b, and the constructed clone product was transformed into E.coli TG1 electroporation competent Cells were used to construct a VHH gene library; the library capacity was determined to be 2.2×10 9 pfu by the plate gradient dilution method, and the colony PCR results showed that the library insertion rate was 97.9%. Take live cells with 10-100 times the library capacity from the above-mentioned gene library for inoculation and culture, and use M13K07 phage to rescue after culturing to the logarithmic phase. After the rescue culture, collect the phage by centrifugation, and use PEG-NaCl to purify the phage to obtain phage display. Libraries can be used directly for subsequent screening.
实施例3 抗人Siglec15抗原结合蛋白的筛选Example 3 Screening of anti-human Siglec15 antigen-binding protein
将重组人Siglec15-His(SG5-H52H3，ACRO)和重组猴Siglec15-His(SG5-C52H6，ACRO)包被到96孔板中，通过Elisa实验筛选3-5轮，将特异结合人和猴Siglec15的噬菌体逐步得到富集。挑选大量阳性克隆进行Elisa检测并对阳性克隆筛选和测序，根据序列比对确定独特的克隆并将其序列分为框架区FR和互补决定区CDR。通过以上方法，获得亲和力较高的靶向人和猴Siglec15的单域抗体(sdAb)。Coat recombinant human Siglec15-His (SG5-H52H3, ACRO) and recombinant monkey Siglec15-His (SG5-C52H6, ACRO) into 96-well plates, and screen for 3-5 rounds through Elisa experiments, and specifically bind to human and monkey Siglec15 The phages were gradually enriched. A large number of positive clones were selected for Elisa detection, and the positive clones were screened and sequenced. The unique clones were determined according to sequence alignment and their sequences were divided into framework region FR and complementarity determining region CDR. Through the above method, a single-domain antibody (sdAb) targeting human and monkey Siglec15 with high affinity was obtained.
实施例4 人源化Siglec15抗原结合蛋白Example 4 Humanized Siglec15 antigen-binding protein
为了降低驼源抗体的免疫原性，将已筛选出的有生物学活性的抗体进行人源化。驼源单克隆抗体的人源化根据本领域许多文献公示的方法进行。可以使用人抗体恒定结构域替代亲本(驼源抗体)恒定结构域，根据驼源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。或者可以以驼源抗体的三维结构为基础，通过驼源抗体VH的氨基酸残基进行回复突变，将驼源抗体的恒定区替换为人恒定区，得到最终的人源化结合蛋白。本实施例人源化后的抗原结合蛋白如表1所示。In order to reduce the immunogenicity of camel-derived antibodies, the screened biologically active antibodies were humanized. The humanization of camel monoclonal antibodies is carried out according to the methods published in many documents in this field. Human antibody constant domains can be used to replace the parental (camel antibody) constant domains, and human germline antibody sequences can be selected according to the homology between camel antibodies and human antibodies for CDR grafting. Alternatively, based on the three-dimensional structure of the camel antibody, the constant region of the camel antibody can be replaced by a human constant region through back-mutation of the amino acid residues of the VH of the camel antibody to obtain the final humanized binding protein. The antigen-binding proteins after humanization in this example are shown in Table 1.
表1 人源化后的抗原结合蛋白的可变区序列信息Table 1 The variable region sequence information of the humanized antigen-binding protein
Figure PCTCN2022115816-appb-000001
Figure PCTCN2022115816-appb-000001
Figure PCTCN2022115816-appb-000002
Figure PCTCN2022115816-appb-000002
实施例5 Siglec15抗原结合蛋白的表达和纯化Example 5 Expression and purification of Siglec15 antigen-binding protein
根据实施例4得到的抗原结合蛋白序列构建质粒，瞬转表达Expi293细胞(Thermo Fisher，货号：A14527CN)，使用大量抽提试剂盒提取抗体表达质粒。溶液1：用1ml培养液稀释15μg 质粒，混匀。溶液2：用1ml培养液稀释60μl转染试剂，混匀。将溶液2加入溶液1中，混匀，37℃孵育15分钟后，将混合转染液逐滴加入细胞液中，边摇边加，放至摇床培养，表达一周，收集上清，8000转/分钟离心5分钟。A plasmid was constructed according to the sequence of the antigen-binding protein obtained in Example 4, which was transiently expressed in Expi293 cells (Thermo Fisher, catalog number: A14527CN), and the antibody expression plasmid was extracted using a large-scale extraction kit. Solution 1: Dilute 15μg plasmid with 1ml culture medium and mix well. Solution 2: Dilute 60μl transfection reagent with 1ml culture medium and mix well. Add solution 2 to solution 1, mix well, and incubate at 37°C for 15 minutes, add the mixed transfection solution dropwise to the cell solution, add while shaking, put it on a shaker for culture, express for one week, collect the supernatant, 8000 rpm /min centrifuge for 5 minutes.
Protein A亲和层析柱纯化抗体蛋白：Protein A affinity chromatography column purification of antibody protein:
(1)平衡层析柱：1×PBS，流速1ml/分钟，20ml(1) Equilibrium chromatography column: 1×PBS, flow rate 1ml/min, 20ml
(2)上样：流速1ml/分钟(2) Sample loading: flow rate 1ml/min
(3)洗杂：1×PBS，流速1ml/分钟，20ml(3) Washing: 1×PBS, flow rate 1ml/min, 20ml
(4)洗脱：柠檬酸缓冲液(PH3.4)，1ml/分钟，分管收集，每管约500μl。共收集10管，使用NanoDrop仪器读取280nm吸光度值。(4) Elution: citric acid buffer (PH3.4), 1 ml/min, collected in separate tubes, about 500 μl per tube. A total of 10 tubes were collected and the absorbance value at 280nm was read using a NanoDrop instrument.
(5)透析：将高浓度蛋白吸至透析袋放1×PBS的烧杯中透析。使用高效液相色谱仪LC-20AT及凝胶色谱柱的纯度检测合格，内毒素检测合格。(5) Dialysis: suck the high-concentration protein into a dialysis bag and put it in a beaker of 1×PBS for dialysis. The purity test using high performance liquid chromatography LC-20AT and gel chromatographic column is qualified, and the endotoxin test is qualified.
实施例6 Siglec15抗原结合蛋白与表达于细胞上的人Siglec15的结合Example 6 Binding of Siglec15 antigen-binding protein to human Siglec15 expressed on cells
基于流式细胞术测定法测定实施例5中得到的Ab0(氨基酸序列如SEQ ID NO:8所示)Siglec15抗原结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合能力。通过比较不同Siglec15抗原结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合曲线来测定其结合能力。The binding ability of the Siglec15 antigen-binding protein of Ab0 (amino acid sequence shown in SEQ ID NO: 8) obtained in Example 5 to human Siglec15 expressed on the surface of CHOK1 cells was determined based on flow cytometry. The binding ability of different Siglec15 antigen-binding proteins was determined by comparing the binding curves of human Siglec15 expressed on the surface of CHOK1 cells.
1、将CHOK1/hSiglec15细胞(康源博创)铺于96孔板中。1. Spread CHOK1/hSiglec15 cells (Kangyuan Bochuang) in a 96-well plate.
2、用含2％FBS的PBS分别配制阳性对照抗体(阳性对照抗体，泰州市百英生物科技有限公司，货号B218501)、阴性对照抗体(人IgG1，novoprotein，货号NC002)和本申请抗原结合蛋白最大浓度为15μg/ml，5倍稀释，8个点，稀释好的样品加入96孔板中，4摄氏度孵育1小时。2. Use PBS containing 2% FBS to prepare positive control antibody (positive control antibody, Taizhou Baiying Biotechnology Co., Ltd., product number B218501), negative control antibody (human IgG1, novoprotein, product number NC002) and the antigen-binding protein of this application, respectively. The maximum concentration is 15 μg/ml, 5-fold dilution, 8 points, the diluted sample is added to a 96-well plate, and incubated at 4 degrees Celsius for 1 hour.
3、用含2％FBS的PBS洗板3次。3. Wash the plate 3 times with PBS containing 2% FBS.
4、将PE标记的人IgG(Biolegend,货号410708)分别用2％FBS的PBS按产品说明书比例稀释，稀释好的样品加入96孔板中，4℃度孵育0.5小时。4. Dilute PE-labeled human IgG (Biolegend, Cat. No. 410708) with 2% FBS in PBS according to the product instructions, add the diluted samples to a 96-well plate, and incubate at 4°C for 0.5 hours.
5、用含2％FBS的PBS洗板2次。5. Wash the plate twice with PBS containing 2% FBS.
6、用2％FBS的PBS重新悬浮细胞，使用流式细胞仪测定PE通道的中位荧光度值(MFI)。6. The cells were resuspended in PBS with 2% FBS, and the median fluorescence value (MFI) of the PE channel was measured by flow cytometry.
以上试验表明，本申请中实施例5得到的的Siglec15抗原结合蛋白具有与CHOK1细胞上过表达的人Siglec15的结合活性，并且优于阳性抗体。基于流式细胞术测定Siglec15抗原结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合曲线结果如图1所示。基于流式细胞术测定Siglec15抗原结合蛋白与表达于CHOK1细胞表面的人Siglec15结合的EC50值和 最高MFI值结果如表2所示。The above tests show that the Siglec15 antigen-binding protein obtained in Example 5 of the present application has binding activity to human Siglec15 overexpressed on CHOK1 cells, and is superior to positive antibodies. The results of the binding curve of Siglec15 antigen-binding protein and human Siglec15 expressed on the surface of CHOK1 cells based on flow cytometry are shown in FIG. 1 . Table 2 shows the EC50 value and the highest MFI value of Siglec15 antigen-binding protein binding to human Siglec15 expressed on the surface of CHOK1 cells based on flow cytometry.
表2 Siglec15抗原结合蛋白与表达于CHOK1细胞表面Siglec15结合的EC50值和最高MFI值Table 2 The EC50 value and the highest MFI value of Siglec15 antigen binding protein binding to Siglec15 expressed on the surface of CHOK1 cells
编号serial number EC 50(μg/ml) EC50 (μg/ml) 最高MFI值Highest MFI value
阳性对照抗体positive control antibody 0.36320.3632 15045.8015045.80
阴性对照抗体negative control antibody -- 631.70631.70
Ab0Ab0 0.08460.0846 9062.809062.80
实施例7 Siglec15抗原结合蛋白的生物学活性Example 7 Biological activity of Siglec15 antigen-binding protein
7.1 CD4和CD8T细胞增殖检测7.1 Detection of CD4 and CD8 T cell proliferation
7.1.1、将100μl的OKT3(novoprotein，货号GMP-A018)加入96孔细胞板(Corning，3599)中，4℃孵育过夜。包被结束后用PBS洗三次，待用。7.1.1. Add 100 μl of OKT3 (novoprotein, Cat. No. GMP-A018) to a 96-well cell plate (Corning, 3599), and incubate overnight at 4°C. After coating, wash three times with PBS and set aside.
7.1.2、用PBS稀释待测抗体(Siglec15结合蛋白)终浓度为15μg/ml，同时稀释Siglec15抗原终浓度为5μg/ml，稀释好的样品加入96孔板中。将细胞板放于37℃孵育30min。7.1.2. Dilute the antibody to be tested (Siglec15-binding protein) with PBS to a final concentration of 15 μg/ml, and at the same time dilute the Siglec15 antigen to a final concentration of 5 μg/ml, and add the diluted sample to a 96-well plate. The cell plate was incubated at 37°C for 30min.
7.1.3、冻存的人外周单个核细胞(PBMC)，在37℃水浴锅中化冻后，重悬于PBS中，400g，5min离心弃去上清。将细胞重悬于PBS中调整细胞密度，加入0.5μM CFSE(Invitrogen,货号65-0850-85)，并在室温下孵育8分钟，然后加入等体积的血清，室温下孵育8分钟，400g，5min离心弃去上清。7.1.3. Thaw frozen human peripheral mononuclear cells (PBMC) in a 37°C water bath, resuspend in PBS, centrifuge at 400g for 5 minutes, and discard the supernatant. Resuspend the cells in PBS to adjust the cell density, add 0.5μM CFSE (Invitrogen, product number 65-0850-85), and incubate at room temperature for 8 minutes, then add an equal volume of serum, incubate at room temperature for 8 minutes, 400g, 5min Centrifuge and discard supernatant.
7.1.4、用PBS洗细胞，洗三次。用带血清1640培养基重悬PBMC后，加入到孵育后的抗体孔中，37℃孵育64h。孵育结束后，收集细胞上清液备用。7.1.4. Wash the cells with PBS for three times. PBMCs were resuspended in 1640 medium with serum, added to the incubated antibody wells, and incubated at 37°C for 64h. After incubation, the cell supernatant was collected for use.
7.1.5、用FACS缓冲液(PBS+2％FBS)将细胞反应板中的PBMC转移到96孔U型板中(Corning，货号3799)，400g，5min离心弃去上清。按照说明书用FACS缓冲液稀释APC抗人CD8(BioLegend，344722)和PE-Cy7抗人CD4(BioLegend，357410)抗体，稀释好的样品加入96孔圆底板中，室温孵育30min。400g，5min离心弃去上清。7.1.5. Use FACS buffer (PBS+2% FBS) to transfer the PBMC in the cell reaction plate to a 96-well U-shaped plate (Corning, Cat. No. 3799), centrifuge at 400 g for 5 min, and discard the supernatant. According to the instructions, APC anti-human CD8 (BioLegend, 344722) and PE-Cy7 anti-human CD4 (BioLegend, 357410) antibodies were diluted with FACS buffer, and the diluted samples were added to a 96-well round bottom plate and incubated at room temperature for 30 min. Centrifuge at 400g for 5min and discard the supernatant.
7.1.6、用FACS缓冲液重复洗细胞3次。PBS重悬细胞后流式细胞仪检测，用Kaluza分析数据。数据显示的是基于CFSE含量降低的CD4和CD8T细胞与未刺激的CD4和CD8T细胞的比例。7.1.6. Wash the cells three times with FACS buffer. Cells were resuspended in PBS and detected by flow cytometry, and the data were analyzed by Kaluza. Data shown are based on the ratio of CD4 and CD8 T cells with reduced CFSE content to unstimulated CD4 and CD8 T cells.
7.2用人IFN-g Elisa Kit(RnD,货号DY285B)检测培养基上清中IFN-γ的表达量7.2 Use human IFN-g Elisa Kit (RnD, Cat. No. DY285B) to detect the expression of IFN-γ in the medium supernatant
7.2.1、用PBS将捕获抗体稀释至2μg/ml，按100μl/孔的体积包被96孔板，4℃包被过夜。7.2.1. Dilute the capture antibody to 2 μg/ml with PBS, coat a 96-well plate at a volume of 100 μl/well, and coat overnight at 4°C.
7.2.2、包被结束后用PBST(0.05％
Figure PCTCN2022115816-appb-000003
20in PBS,pH 7.2-7.4)洗三次，洗板后将96 孔板倒扣在洁净的实验室用纸巾上，轻拍若干次，去除孔中残余液体。 7.2.2. Use PBST (0.05%
Figure PCTCN2022115816-appb-000003
20in PBS, pH 7.2-7.4) and washed three times. After washing the plate, place the 96-well plate upside down on a clean laboratory paper towel, and pat it several times to remove the residual liquid in the well.
7.2.3、用含1％BSA的PBST进行封闭，300μl/孔，室温封闭2h。封闭结束后，用PBST洗三次。7.2.3. Block with PBST containing 1% BSA, 300 μl/well, and block for 2 hours at room temperature. After blocking, wash three times with PBST.
7.2.4、用含1％BSA的PBST稀释样品及标准品到使用浓度，按100μl/孔将样品及标准品加入96孔板中，完成后盖上板盖，密封接口处缝隙，室温孵育2h。孵育结束后，用PBST洗三次。7.2.4. Dilute the samples and standards with PBST containing 1% BSA to the use concentration, add the samples and standards to the 96-well plate at 100 μl/well, cover the plate after completion, seal the gap at the interface, and incubate at room temperature for 2 hours . After incubation, wash three times with PBST.
7.2.5、用含1％BSA的PBST将检测抗体稀释至200ng/ml，按100μl/孔将稀释好的检测抗体加入96孔板中，盖上板盖，密封接口处缝隙，室温孵育2h。孵育结束后，用PBST洗三次。7.2.5. Dilute the detection antibody to 200 ng/ml with PBST containing 1% BSA, add the diluted detection antibody to a 96-well plate at 100 μl/well, cover the plate, seal the gap at the interface, and incubate at room temperature for 2 hours. After incubation, wash three times with PBST.
7.2.6、用含1％BSA的PBST按1:40的比例稀释链霉亲和素-HRP至工作浓度，然后将其加入96孔板中，100μl/孔，37℃孵育20min(稀释及孵育时要注意避光)。孵育结束后，用PBST洗三次。7.2.6. Dilute streptavidin-HRP to the working concentration with PBST containing 1% BSA at a ratio of 1:40, then add it to a 96-well plate, 100 μl/well, and incubate at 37°C for 20 minutes (dilution and incubation Be careful to avoid light). After incubation, wash three times with PBST.
7.2.7、将TMB混合液按100μl/孔加入96孔板，37℃孵育15～20min(此步骤需避光操作)。7.2.7. Add 100 μl/well of the TMB mixture into a 96-well plate, and incubate at 37°C for 15-20 minutes (this step needs to be protected from light).
7.2.8、反应结束后，加入50μl终止液反应，轻拍96孔板使其充分混匀。终止反应后，立即用酶标仪读取96孔板在450nm处的吸光度值。通过标准品计算样品中INF-γ的水平。7.2.8. After the reaction is over, add 50 μl of stop solution to react, and tap the 96-well plate to mix well. Immediately after terminating the reaction, read the absorbance value of the 96-well plate at 450 nm with a microplate reader. The level of INF-γ in the samples was calculated from the standards.
以上试验表明，本申请Siglec15抗原结合蛋白能够阻断Siglec15对受试者血液中PBMC的增殖抑制能力。实验结果如图2A-C所示，Siglec15抗原结合蛋白能够促进CD8和CD4 T细胞的增殖及促进培养基上清中IFN-γ的表达量性，且活性与阳性抗体相当或优于阳性抗体。实验结果如表3所示，Siglec15抗原结合蛋白能够阻断Siglec15对受试者血液中PBMC的增殖抑制能力。The above tests show that the Siglec15 antigen-binding protein of the present application can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the blood of subjects. The experimental results are shown in Figure 2A-C, Siglec15 antigen-binding protein can promote the proliferation of CD8 and CD4 T cells and the expression of IFN-γ in the culture supernatant, and its activity is equivalent to or better than that of the positive antibody. The experimental results are shown in Table 3. The Siglec15 antigen binding protein can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the subject's blood.
表3 Siglec15结合阻断Siglec15对受试者血液中PBMC的增殖抑制能力Table 3 The ability of Siglec15 binding to block Siglec15 to inhibit the proliferation of PBMC in the blood of subjects
Figure PCTCN2022115816-appb-000004
Figure PCTCN2022115816-appb-000004
Figure PCTCN2022115816-appb-000005
Figure PCTCN2022115816-appb-000005
实施例8 人源化Siglec15结合蛋白与表达于细胞上的人Siglec15的结合Example 8 Binding of humanized Siglec15-binding protein to human Siglec15 expressed on cells
基于流式细胞术测定法测定Siglec15结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合能力。通过比较本申请不同人源化Siglec15结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合曲线来测定其结合能力。The binding ability of Siglec15-binding protein to human Siglec15 expressed on the surface of CHOK1 cells was determined based on a flow cytometry assay. The binding ability of different humanized Siglec15 binding proteins of the present application was determined by comparing the binding curves of human Siglec15 expressed on the surface of CHOK1 cells.
1、将CHOK1/hSiglec15细胞(康源博创)铺于96孔板中。1. Spread CHOK1/hSiglec15 cells (Kangyuan Bochuang) in a 96-well plate.
2、用含2％FBS的PBS分别配制阳性对照抗体(SPH-Sg-PC-1，泰州市百英生物科技有限公司，货号B218501)、阴性对照抗体(人IgG1，novoprotein，货号NC002)和本申请抗原结合蛋白最大浓度为15μg/ml，5倍稀释，8个点，稀释好的样品加入96孔板中，4摄氏度孵育1小时。2. Use PBS containing 2% FBS to prepare positive control antibody (SPH-Sg-PC-1, Taizhou Baiying Biotechnology Co., Ltd., product number B218501), negative control antibody (human IgG1, novoprotein, product number NC002) and this Apply for a maximum concentration of antigen-binding protein of 15 μg/ml, 5-fold dilution, 8 points, add the diluted sample to a 96-well plate, and incubate at 4 degrees Celsius for 1 hour.
3、用含2％FBS的PBS洗板3次。3. Wash the plate 3 times with PBS containing 2% FBS.
4、将PE标记的-人IgG(Biolegend,货号410708)分别用2％FBS的PBS按产品说明书比例稀释，稀释好的样品加入96孔板中，4摄氏度孵育0.5小时。4. Dilute PE-labeled human IgG (Biolegend, Cat. No. 410708) with 2% FBS in PBS according to the product instructions, add the diluted samples to a 96-well plate, and incubate at 4°C for 0.5 hours.
5、用含2％FBS的PBS洗板2次。5. Wash the plate twice with PBS containing 2% FBS.
6、用2％FBS的PBS重新悬浮细胞，使用流式细胞仪测定PE通道的中位荧光度值(MFI)。6. The cells were resuspended in PBS with 2% FBS, and the median fluorescence value (MFI) of the PE channel was measured by flow cytometry.
在以上试验表明，本申请的人源化Siglec15结合蛋白具有与CHOK1细胞上过表达的人Siglec15的结合活性，并且优于阳抗。实验结果如图3A-B所示，基于流式细胞术测定人源化Siglec15结合蛋白与表达于CHOK1细胞表面的人Siglec15的结合曲线。实验结果如表4所示，基于流式细胞术测定人源化Siglec15结合蛋白与表达于CHOK1细胞表面的人Siglec15结合的EC 50值和最高MFI值。 The above tests show that the humanized Siglec15-binding protein of the present application has binding activity to human Siglec15 overexpressed on CHOK1 cells, and is superior to positive antibodies. The experimental results are shown in Figure 3A-B, the binding curve of humanized Siglec15 binding protein and human Siglec15 expressed on the surface of CHOK1 cells was determined based on flow cytometry. The experimental results are shown in Table 4. The EC50 value and the highest MFI value of the humanized Siglec15-binding protein binding to human Siglec15 expressed on the surface of CHOK1 cells were determined based on flow cytometry.
表4 人源化Siglec15结合蛋白与表达于CHOK1细胞表面Siglec15结合的EC 50值和最高MFI值 Table 4 EC 50 value and highest MFI value of humanized Siglec15-binding protein binding to Siglec15 expressed on the surface of CHOK1 cells
编号serial number EC 50(μg/ml) EC50 (μg/ml) 最高MFI值Highest MFI value
阳性对照抗体positive control antibody 0.12760.1276 194279.00194279.00
阴性对照抗体negative control antibody ～~ 4206.004206.00
Ab0Ab0 0.04770.0477 265240.50265240.50
Ab1Ab1 0.00730.0073 164974.00164974.00
Ab2Ab2 0.01540.0154 178793.00178793.00
Ab3Ab3 0.01050.0105 202322.00202322.00
Ab4Ab4 0.00180.0018 169629.00169629.00
Ab5Ab5 0.02860.0286 215818.50215818.50
Ab6Ab6 0.21390.2139 140492.50140492.50
Ab7Ab7 0.21180.2118 193694.00193694.00
Ab8Ab8 0.12010.1201 158883.00158883.00
Ab9Ab9 0.06800.0680 199439.00199439.00
Ab10Ab10 0.01650.0165 151266.00151266.00
Ab11Ab11 0.01570.0157 164772.00164772.00
实施例9 人源化Siglec15结合蛋白的生物学活性Example 9 Biological activity of humanized Siglec15 binding protein
9.1 CD4和CD8T细胞增殖检测9.1 Detection of CD4 and CD8 T cell proliferation
9.1.1、将100μl的OKT3(novoprotein，货号GMP-A018)加入96孔细胞板(Corning，3599)中，4℃孵育过夜。包被结束后用PBS洗三次，待用。9.1.1. Add 100 μl of OKT3 (novoprotein, Cat. No. GMP-A018) to a 96-well cell plate (Corning, 3599), and incubate overnight at 4°C. After coating, wash three times with PBS and set aside.
9.1.2、用PBS稀释待测抗体(Siglec15结合蛋白)终浓度为15μg/ml，，同时稀释Siglec15抗原终浓度为5μg/ml，稀释好的样品加入96孔板中。将细胞板放于37℃孵育30min。9.1.2. Dilute the antibody to be tested (Siglec15-binding protein) with PBS to a final concentration of 15 μg/ml, and at the same time dilute the Siglec15 antigen to a final concentration of 5 μg/ml, and add the diluted sample to a 96-well plate. The cell plate was incubated at 37°C for 30min.
9.1.3、冻存的人外周单个核细胞(PBMC)，在37℃水浴锅中化冻后，重悬于PBS中，400g，5min离心弃去上清。将细胞重悬于PBS中调整细胞密度，加入0.5μM CFSE(Invitrogen,货号65-0850-85)，并在室温下孵育8分钟，然后加入等体积的血清，室温下孵育8分钟，400g，5min离心弃去上清。9.1.3. Thaw frozen human peripheral mononuclear cells (PBMC) in a 37°C water bath, resuspend in PBS, centrifuge at 400g for 5 minutes, and discard the supernatant. Resuspend the cells in PBS to adjust the cell density, add 0.5μM CFSE (Invitrogen, product number 65-0850-85), and incubate at room temperature for 8 minutes, then add an equal volume of serum, incubate at room temperature for 8 minutes, 400g, 5min Centrifuge and discard supernatant.
9.1.4、用PBS洗细胞，洗三次。用带血清1640培养基重悬PBMC后，加入到孵育后的抗体孔中，37℃孵育64h。孵育结束后，收集细胞上清液备用。9.1.4. Wash the cells with PBS for three times. PBMCs were resuspended in 1640 medium with serum, added to the incubated antibody wells, and incubated at 37°C for 64h. After incubation, the cell supernatant was collected for use.
9.1.5、用FACS缓冲液(PBS+2％FBS)将细胞反应板中的PBMC转移到96孔U型板中(Corning，货号3799)，400g，5min离心弃去上清。按照说明书用FACS缓冲液稀释APC抗人CD8(BioLegend，344722)和PE-Cy7抗人CD4(BioLegend，357410)抗体，稀释好的样品加入96孔圆底板中，室温孵育30min。400g，5min离心弃去上清。9.1.5. Use FACS buffer (PBS+2% FBS) to transfer the PBMCs in the cell reaction plate to a 96-well U-shaped plate (Corning, Cat. No. 3799), centrifuge at 400 g for 5 min, and discard the supernatant. According to the instructions, APC anti-human CD8 (BioLegend, 344722) and PE-Cy7 anti-human CD4 (BioLegend, 357410) antibodies were diluted with FACS buffer, and the diluted samples were added to a 96-well round bottom plate and incubated at room temperature for 30 min. Centrifuge at 400g for 5min and discard the supernatant.
9.1.6、用FACS缓冲液重复洗细胞3次。PBS重悬细胞后流式细胞仪检测，用Kaluza分析数据。数据显示的是基于CFSE含量降低的CD4和CD8T细胞与未刺激的CD4和CD8T细胞的比例。9.1.6. Repeat washing the cells 3 times with FACS buffer. Cells were resuspended in PBS and detected by flow cytometry, and the data were analyzed by Kaluza. Data shown are based on the ratio of CD4 and CD8 T cells with reduced CFSE content to unstimulated CD4 and CD8 T cells.
9.2用人IFN-g Elisa Kit(RnD,货号DY285B)检测培养基上清中IFN-γ的表达量9.2 Use human IFN-g Elisa Kit (RnD, Cat. No. DY285B) to detect the expression of IFN-γ in the medium supernatant
9.2.1、用PBS将捕获抗体稀释至2μg/ml，按100μl/孔的体积包被96孔板，4℃包被过夜。9.2.1. Dilute the capture antibody to 2 μg/ml with PBS, coat a 96-well plate at a volume of 100 μl/well, and coat overnight at 4°C.
9.2.2、包被结束后用PBST(0.05％
Figure PCTCN2022115816-appb-000006
20in PBS,pH 7.2-7.4)洗三次，洗板后将96孔板倒扣在洁净的实验室用纸巾上，轻拍若干次，去除孔中残余液体。 9.2.2. Use PBST (0.05%
Figure PCTCN2022115816-appb-000006
20in PBS, pH 7.2-7.4) and washed three times. After washing the plate, place the 96-well plate upside down on a clean laboratory paper towel, and pat it several times to remove the residual liquid in the well.
9.2.3、用含1％BSA的PBST进行封闭，300μl/孔，室温封闭2h。封闭结束后，用PBST洗三次。9.2.3. Block with PBST containing 1% BSA, 300 μl/well, and block for 2 hours at room temperature. After blocking, wash three times with PBST.
9.2.4、用含1％BSA的PBST稀释样品及标准品到使用浓度，按100μl/孔将样品及标准品加入96孔板中，完成后盖上板盖，密封接口处缝隙，室温孵育2h。孵育结束后，用PBST洗三次。9.2.4. Dilute the samples and standards with PBST containing 1% BSA to the use concentration, add the samples and standards to the 96-well plate at 100 μl/well, cover the plate after completion, seal the gap at the interface, and incubate at room temperature for 2 hours . After incubation, wash three times with PBST.
9.2.5、用含1％BSA的PBST将检测抗体稀释至200ng/ml，按100μl/孔将稀释好的检测抗体加入96孔板中，盖上板盖，密封接口处缝隙，室温孵育2h。孵育结束后，用PBST洗三次。9.2.5. Dilute the detection antibody to 200 ng/ml with PBST containing 1% BSA, add the diluted detection antibody to the 96-well plate at 100 μl/well, cover the plate, seal the gap at the interface, and incubate at room temperature for 2 hours. After incubation, wash three times with PBST.
9.2.6、用含1％BSA的PBST按1:40的比例稀释链霉亲和素-HRP至工作浓度，然后将其加入96孔板中，100μl/孔，37℃孵育20min(稀释及孵育时要注意避光)。孵育结束后，用PBST洗三次。9.2.6. Dilute streptavidin-HRP with PBST containing 1% BSA at a ratio of 1:40 to the working concentration, then add it to a 96-well plate, 100 μl/well, and incubate at 37°C for 20 minutes (dilution and incubation Be careful to avoid light). After incubation, wash three times with PBST.
9.2.7、将TMB混合液按100μl/孔加入96孔板，37℃孵育15～20min(此步骤需避光操作)。9.2.7. Add 100 μl/well of the TMB mixture into a 96-well plate, and incubate at 37°C for 15-20 minutes (this step needs to be protected from light).
9.2.8、反应结束后，加入50μl终止液反应，轻拍96孔板使其充分混匀。终止反应后，立即用酶标仪读取96孔板在450nm处的吸光度值。通过标准品计算样品中INF-γ的水平。9.2.8. After the reaction is over, add 50 μl of stop solution to react, and tap the 96-well plate to mix well. Immediately after terminating the reaction, read the absorbance value of the 96-well plate at 450 nm with a microplate reader. The level of INF-γ in the samples was calculated from the standards.
以上试验表明，本申请人源化Siglec15抗原结合蛋白能够阻断Siglec15对受试者血液中PBMC的增殖抑制能力。实验结果如图4A-C所示，人源化Siglec15抗原结合蛋白能够促进CD8和CD4T细胞的增殖以及促进培养基上清中IFN-γ的表达量性，并且活性优于阳性抗体。实验结果如表5所示，Siglec15抗原结合蛋白能够阻断Siglec15对受试者血液中PBMC的增殖抑制能力。The above experiments show that the humanized Siglec15 antigen-binding protein of the present application can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the subject's blood. The experimental results are shown in Figure 4A-C, the humanized Siglec15 antigen-binding protein can promote the proliferation of CD8 and CD4 T cells and the quantitative expression of IFN-γ in the culture supernatant, and its activity is better than that of the positive antibody. The experimental results are shown in Table 5. The Siglec15 antigen-binding protein can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the subject's blood.
表5 Siglec15抗原结合蛋白阻断Siglec15对受试者血液中PBMC的增殖抑制能力Table 5 Siglec15 antigen-binding protein blocks the ability of Siglec15 to inhibit the proliferation of PBMC in the blood of subjects
Figure PCTCN2022115816-appb-000007
Figure PCTCN2022115816-appb-000007
Figure PCTCN2022115816-appb-000008
Figure PCTCN2022115816-appb-000008
实施例10 人源化Siglec15抗原结合蛋白的结合亲和力Example 10 Binding affinity of humanized Siglec15 antigen-binding protein
用octet RED 384检测不同Siglec15抗原结合蛋白与抗原(Siglec15蛋白，人，重组(ECD,His Tag)，来源:Acro，货号:SG5-C5253)蛋白的结合亲和性。 The binding affinity of different Siglec15 antigen-binding proteins to antigen (Siglec15 protein, human, recombinant (ECD, His Tag), source: Acro, catalog number: SG5-C5253) protein was detected by octet RED 384.
1、试剂准备。Buffer配制：100mlPBS(gibco,货号10010-023)加入20ul Tween 20(SIGMA，货号P1379-500ML)，以每孔220μl加入到Bio-One 96-well black flat-bottom孔板(Greiner，货号655209)的相应位置中。再生液：以Glycine溶液(10mM,PH 1.7)每孔220μl加入到Bio-One 96-well black flat-bottom孔板的相应位置中。1. Reagent preparation. Buffer preparation: 100mlPBS (gibco, catalog number 10010-023) was added to 20ul Tween 20 (SIGMA, catalog number P1379-500ML), and 220μl per well was added to the well plate of Bio-One 96-well black flat-bottom (Greiner, catalog number 655209). in the corresponding position. Regeneration Solution: Add 220μl per well of Glycine solution (10mM, PH 1.7) to the corresponding position of the Bio-One 96-well black flat-bottom well plate.
2、样品配制。以PBST(0.02％Tween20)将阳性对照抗体，阴性待测抗体和待测Siglec15抗原结合蛋白稀释为5μg/ml，每个Siglec15抗原结合蛋白设置实验组和参照组，以每孔220μl加入到Bio-One 96-well black flat-bottom孔板的相应位置中。2. Sample preparation. Dilute the positive control antibody, negative antibody to be tested and Siglec15 antigen-binding protein to be tested with PBST (0.02% Tween20) to 5 μg/ml, set up experimental group and reference group for each Siglec15 antigen-binding protein, add 220 μl per well to Bio- In the corresponding position of the One 96-well black flat-bottom orifice plate.
3、抗原配制。以PBST(0.02％Tween20)将human Siglec15-His稀释为100nM，，以每孔220μl加入到Bio-One 96-well black flat-bottom孔板的相应位置中。3. Antigen preparation. Dilute human Siglec15-His to 100nM with PBST (0.02% Tween20), and add 220 μl per well to the corresponding position of the Bio-One 96-well black flat-bottom well plate.
4、取新的Bio-One 96-well black flat-bottom孔板放置于octet RED 384左侧托架上，其上放置绿色托盘，托盘第一列放置AHC Dip and Read Biosensors(ForteBio，货号18-5060)。octet RED 384中间或右侧托架上放置配制样品的Bio-One 96-well black flat-bottom孔板。 4. Take a new Bio-One 96-well black flat-bottom orifice plate and place it on the left side bracket of octet RED 384, place a green tray on it, and place AHC Dip and Read Biosensors (ForteBio, Cat. No. 18- 5060). The middle or right rack of the octet RED 384 is placed on the Bio-One 96-well black flat-bottom well plate for preparing samples.
5、上机检测分析。以PBST(0.02％Tween20)baseline 60,将待测阳性对照抗体，阴性待测抗体和待测Siglec15抗原结合蛋白固定120s,结合180秒，解离180秒，每一个Siglec15抗原结合蛋白分析后，芯片需要用Glycine溶液再生30s，洗掉抗原结合蛋白以及未解离的分析物。使用Data Analysis 12.0分析软件计算每个抗体的K D值。对照组用于背景的扣减。 5. On-board testing and analysis. Using PBST (0.02% Tween20) baseline 60, fix the positive control antibody to be tested, the negative antibody to be tested and the Siglec15 antigen-binding protein to be tested for 120s, combine for 180 seconds, and dissociate for 180 seconds. After each Siglec15 antigen-binding protein is analyzed, the chip It needs to be regenerated with Glycine solution for 30s to wash away the antigen binding protein and undissociated analyte. The KD value of each antibody was calculated using Data Analysis 12.0 analysis software. The control group was used for background subtraction.
以上试验表明，本申请人源化Siglec15抗原结合蛋白具有与抗原的结合亲和力。The above tests show that the humanized Siglec15 antigen-binding protein of the present application has binding affinity with antigen.
实验结果如表6所示，用Biacore检测不同人源化Siglec15抗原结合蛋白与抗原蛋白的结合亲和性。The experimental results are shown in Table 6. Biacore was used to detect the binding affinities of different humanized Siglec15 antigen-binding proteins to antigen proteins.
表6 Siglec15抗原结合蛋白与抗原蛋白的结合亲和力Table 6 Binding affinity of Siglec15 antigen binding protein and antigen protein
编号serial number ka(1/Ms)ka(1/Ms) kd(1/s)kd(1/s) K D(M) K D (M)
阳性对照抗体positive control antibody 5.15E+055.15E+05 2.49E-042.49E-04 4.84E-104.84E-10
Ab0Ab0 1.93E+051.93E+05 2.17E-042.17E-04 1.13E-091.13E-09
Ab1Ab1 1.75E+051.75E+05 3.18E-043.18E-04 1.82E-091.82E-09
Ab2Ab2 1.84E+051.84E+05 3.21E-043.21E-04 1.75E-091.75E-09
Ab3Ab3 2.03E+052.03E+05 4.00E-044.00E-04 1.98E-091.98E-09
Ab4Ab4 1.92E+051.92E+05 3.95E-043.95E-04 2.06E-092.06E-09
Ab5Ab5 1.50E+051.50E+05 2.64E-042.64E-04 1.76E-091.76E-09
Ab6Ab6 1.96E+051.96E+05 9.13E-049.13E-04 4.66E-094.66E-09
Ab7Ab7 2.02E+052.02E+05 6.39E-046.39E-04 3.17E-093.17E-09
Ab8Ab8 1.92E+051.92E+05 7.94E-047.94E-04 4.13E-094.13E-09
Ab9Ab9 2.01E+052.01E+05 4.38E-044.38E-04 2.18E-092.18E-09
Ab10Ab10 2.19E+052.19E+05 3.83E-043.83E-04 1.75E-091.75E-09
Ab11Ab11 2.14E+052.14E+05 3.74E-043.74E-04 1.75E-091.75E-09
实施例11 人源化Siglec15抗原结合蛋白的种属交叉反应试验Example 11 Species cross-reactivity test of humanized Siglec15 antigen-binding protein
本试验检测Siglec15抗原结合蛋白分别与人Siglec15-his(Acro,货号5G5-H52H3)，食蟹猴Siglec15-hFc(Acro,货号SG5-C5253)，小鼠Siglec15-his(Acro,货号SG5-M52H7)蛋白的结合。This test detects the interaction between Siglec15 antigen binding protein and human Siglec15-his (Acro, Cat. No. 5G5-H52H3), cynomolgus Siglec15-hFc (Acro, Cat. protein binding.
11.1、用PBS分别配制人Siglec15-his，食蟹猴Siglec15-hFc和小鼠Siglec15-his 1ug/ml，加入96孔板中(Corning，货号9018)，4度过夜。11.1. Prepare human Siglec15-his, cynomolgus monkey Siglec15-hFc and mouse Siglec15-his 1ug/ml in PBS respectively, add to 96-well plate (Corning, Cat. No. 9018), and spend 4 nights overnight.
11.2、次日弃掉包被液，用0.05％Tween20的PBS洗板3次。11.2. Discard the coating solution the next day, and wash the plate 3 times with 0.05% Tween20 PBS.
11.3、用含3％BSA的PBS 37度孵育2小时，用0.05％Tween20的DPBS洗板3次。11.3. Incubate with PBS containing 3% BSA at 37 degrees for 2 hours, and wash the plate 3 times with 0.05% Tween20 in DPBS.
11.4、用含1％BSA的PBS分别配制待测抗体最大浓度为50ug/ml，5倍稀释，12个浓度点。加入96孔板中，室温孵育1小时。用0.05％Tween20的PBS洗板3次。11.4. Use PBS containing 1% BSA to prepare the antibody to be tested at a maximum concentration of 50ug/ml, 5-fold dilution, and 12 concentration points. Add to 96-well plate and incubate at room temperature for 1 hour. The plate was washed 3 times with 0.05% Tween20 in PBS.
11.5、将二抗HRP-山羊抗人IgG F(ab')2(Jackson，货号109-006-008)用含1％BSA的PBS按产品说明书稀释，稀释好的样品加入96孔板中,室温孵育0.5小时。用0.05％Tween20的PBS洗板3次。11.5. Dilute the secondary antibody HRP-goat anti-human IgG F(ab')2 (Jackson, Cat. No. 109-006-008) with PBS containing 1% BSA according to the product instructions. Add the diluted samples to a 96-well plate and store at room temperature. Incubate for 0.5 hours. The plate was washed 3 times with 0.05% Tween20 in PBS.
11.6、每孔加入100ul TMB底物显色液，室温孵育15分钟。11.6. Add 100ul TMB substrate chromogenic solution to each well and incubate at room temperature for 15 minutes.
11.7、每孔加入100ul 2N H 2SO 4终止液，用酶标仪在450nm波长处读取数值并记录。 11.7. Add 100ul 2N H 2 SO 4 stop solution to each well, read the value with a microplate reader at a wavelength of 450nm and record it.
以上试验表明，本申请人源化Siglec15抗原结合蛋白具有与人、猴和鼠Siglec15抗原的结合能力。实验结果如图5A-F所示，人源化Siglec15抗原结合蛋白具有与人、猴和鼠Siglec15抗原的结合曲线。实验结果如表7所示，人源化Siglec15抗原结合蛋白与人、猴和鼠Siglec15抗原的结合能力。The above tests show that the humanized Siglec15 antigen-binding protein of the present application has the ability to bind human, monkey and mouse Siglec15 antigens. The experimental results are shown in Figure 5A-F, the humanized Siglec15 antigen binding protein has binding curves to human, monkey and mouse Siglec15 antigens. The experimental results are shown in Table 7, the binding ability of humanized Siglec15 antigen binding protein to human, monkey and mouse Siglec15 antigen.
表7 人源化Siglec15抗原结合蛋白与人、猴和鼠Siglec15抗原的结合能力Table 7 The binding ability of humanized Siglec15 antigen-binding protein to human, monkey and mouse Siglec15 antigen
Figure PCTCN2022115816-appb-000009
Figure PCTCN2022115816-appb-000009
Figure PCTCN2022115816-appb-000010
Figure PCTCN2022115816-appb-000010
实施例12 Siglec15抗原结合蛋白对野生型C57BL/6N动物模型的抗肿瘤药效试验Example 12 Antitumor efficacy test of Siglec15 antigen-binding protein on wild-type C57BL/6N animal model
本试验采用MC38/hSiglec15细胞接种C57BL/6N小鼠测定本发明的Siglec15抗原结合蛋白的抗肿瘤作用。In this experiment, MC38/hSiglec15 cells were used to inoculate C57BL/6N mice to determine the anti-tumor effect of the Siglec15 antigen-binding protein of the present invention.
C57BL/6N小鼠：雌性C57BL/6N小鼠(6周)(浙江维通利华实验动物技术有限公司)。小鼠在到达后适应性饲养7天，随后开始研究。C57BL/6N mice: female C57BL/6N mice (6 weeks) (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.). Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
细胞：鼠源MC38细胞(和元生物技术(上海)股份有限公司)按照说明书进行常规传代培养；SPH对MC38细胞进行了基因改造，使其过量表达人源Siglec15，该细胞被命名为MC38/hSiglec15细胞，用于后续体内实验。离心收集细胞，在无血清培养基中重悬细胞并调整细胞密度，在第0天将细胞悬液皮下接种至野生型C57BL/6N小鼠右腋窝皮下来建立MC38/hSiglec15荷瘤小鼠模型。Cells: mouse MC38 cells (Heyuan Biotechnology (Shanghai) Co., Ltd.) were routinely subcultured according to the instructions; SPH genetically modified MC38 cells to overexpress human Siglec15, and the cells were named MC38/hSiglec15 cells for subsequent in vivo experiments. The cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of wild-type C57BL/6N mice to establish the MC38/hSiglec15 tumor-bearing mouse model.
给药：肿瘤细胞接种第6天，检测各只小鼠瘤体积，挑选出瘤体积在100mm 3～190mm 3范围内的小鼠按瘤体积平均分组(每组6只小鼠)，分别在接种后第6、9、12、15、18、21天给药，阴性对照抗体(人IgG)、阳性对照抗体(SPH-Sg-PC-1，泰州市百英生物科技有限公司，货号B218501)、本申请抗原结合蛋白，给药期间监测各组小鼠瘤体积和体重变化，监测频率均为2次/周，连续监测3周。在每次给药前测定体重和肿瘤体积，接种后第24天计算肿瘤体积抑制率(TGI％)，计算公式如下：TGI(％)＝[1-(T i-T 0)/(V i-V 0)]×100；其中Ti：给药组肿瘤体积均数，T 0：给药组D0天的肿瘤体积均数，V i：同型对照组肿瘤体积均数，V 0：同型对照组D0天的肿瘤体积均数。肿瘤体积测定：采用游标卡尺测定肿瘤的长径(a)和宽径(b)，肿瘤体积按如下公式计算：T V＝1/2×a×b 2。采用电子天平测定体重。给药剂量和方式如表8所示。 Administration: On the 6th day after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with a tumor volume in the range of 100 mm 3 to 190 mm 3 were selected and divided into groups according to the average tumor volume (6 mice in each group). After administration on the 6th, 9th, 12th, 15th, 18th, and 21st day, negative control antibody (human IgG), positive control antibody (SPH-Sg-PC-1, Taizhou Baiying Biotechnology Co., Ltd., catalog number B218501), For the antigen-binding protein of the present application, the tumor volume and weight changes of the mice in each group were monitored during the administration period, and the monitoring frequency was 2 times/week, and the monitoring was continued for 3 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 24th day after inoculation. The calculation formula was as follows: TGI (%)=[1-(T i -T 0 )/(V i -V 0 )]×100; where Ti: average tumor volume of the administration group, T 0 : average tumor volume of the administration group on D0 day, V i : average tumor volume of the same-type control group, V 0 : the same-type control group The average tumor volume on D0 day. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: T V =1/2×a×b 2 . Body weight was measured using an electronic balance. The dosage and method of administration are shown in Table 8.
表8 给药试验设计Table 8 Drug administration trial design
编号serial number 给药剂量Dosage 给药次数Dosing times 给药方式Method of administration
阴性对照抗体negative control antibody 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab0Ab0 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab1Ab1 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab2Ab2 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab3Ab3 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab4Ab4 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
*表示：每隔3天给药，共6次。*Indicates: Dosing every 3 days for a total of 6 doses.
抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4在野生型C57BL/6N小鼠体内的抗肿瘤药效检测实验结果如图6A所示。抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4施用后，野生型C57BL/6N小鼠的体重变化实验结果如图6B所示。接种后第24天，本申请的抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4在10mg/kg剂量下显著性抑制肿瘤的生长，肿瘤体积抑制率为97％、89％、100％、95％和104％，实验结果如图6C所示。本实施例中，第24天的抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4体内抑制肿瘤生长能力如表9所示。上述试验表明，本申请的抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4显著性抑制肿瘤的生长，并且对野生型C57BL/6N小鼠动物体重均没有明显影响。The experimental results of the anti-tumor efficacy of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 in wild-type C57BL/6N mice are shown in Figure 6A. After administration of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4, the experimental results of body weight changes in wild-type C57BL/6N mice are shown in Figure 6B. On the 24th day after inoculation, the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application significantly inhibited the growth of tumors at a dose of 10 mg/kg, and the tumor volume inhibition rates were 97%, 89%, 100%, and 95%. and 104%, the experimental results are shown in Fig. 6C. In this example, the ability of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 to inhibit tumor growth in vivo on day 24 is shown in Table 9. The above experiments show that the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application significantly inhibit the growth of tumors, and have no significant effect on the body weight of wild-type C57BL/6N mice.
表9 第24天本申请的抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4体内抑制肿瘤生长能力Table 9 On the 24th day, the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application inhibit tumor growth in vivo
Figure PCTCN2022115816-appb-000011
Figure PCTCN2022115816-appb-000011
**表示：与阴性对照抗体肿瘤体积相比，P<0.01。** indicates: P<0.01 compared with the negative control antibody tumor volume.
实施例13 Siglec15抗原结合蛋白对人Siglec15转基因小鼠的抗肿瘤药效试验Example 13 Antitumor efficacy test of Siglec15 antigen-binding protein on human Siglec15 transgenic mice
本试验采用MC38/hSiglec15细胞接种人Siglec15转基因小鼠测定本发明的Siglec15抗原结合蛋白的抗肿瘤作用。In this experiment, MC38/hSiglec15 cells were used to inoculate human Siglec15 transgenic mice to determine the anti-tumor effect of the Siglec15 antigen-binding protein of the present invention.
人Siglec15转基因小鼠：雌性C57BL/6背景的人Siglec15转基因小鼠(6周)(上海南方模式生物科技股份有限公司)。小鼠在到达后适应性饲养7天，随后开始研究。Human Siglec15 transgenic mice: female human Siglec15 transgenic mice (6 weeks) of C57BL/6 background (Shanghai Southern Model Biotechnology Co., Ltd.). Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
细胞：鼠源MC38细胞购自和元生物技术(上海)股份有限公司，按照说明书进行常规传代培养；对MC38细胞进行基因改造，使其过量表达人源Siglec15，该细胞被命名为MC38/人Siglec15细胞，用于后续体内实验。离心收集细胞，在无血清培养基中重悬细胞并调整细 胞密度，在第0天将细胞悬液皮下接种至人Siglec15转基因小鼠右腋窝皮下来建立MC38/人Siglec15荷瘤小鼠模型。Cells: Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and conventionally subcultured according to the instructions; MC38 cells were genetically modified to overexpress human Siglec15, and the cells were named MC38/human Siglec15 cells for subsequent in vivo experiments. The cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of human Siglec15 transgenic mice to establish the MC38/human Siglec15 tumor-bearing mouse model.
给药：肿瘤细胞接种第6/5天，检测各只小鼠瘤体积，挑选出瘤体积在80mm 3～150mm 3范围内的小鼠按瘤体积平均分组(每组5～6只小鼠)，分别在接种后第6/5、9/8、12/11、15/14、18/17、21/20天给药，阴性对照抗体、阳性对照抗体(泰州市百英生物科技有限公司，货号B218501)本申请抗原结合蛋白，给药期间监测各组小鼠瘤体积和体重变化，监测频率均为2次/周，连续监测3周。在每次给药前测定体重和肿瘤体积，接种后第25/23天计算肿瘤体积抑制率(TGI％)，计算公式如下：TGI(％)＝[1-(T i-T 0)/(V i-V 0)]×100；其中T i：给药组肿瘤体积均数，T 0：给药组D0天的肿瘤体积均数，Vi：同型对照组肿瘤体积均数，V0：同型对照组D0天的肿瘤体积均数。肿瘤体积测定：采用游标卡尺测定肿瘤的长径(a)和宽径(b)，肿瘤体积按如下公式计算：T V＝1/2×a×b 2。采用电子天平测定体重。给药剂量和方式如表10所示。 Administration: On the 6th/5th day of tumor cell inoculation, detect the tumor volume of each mouse, and select mice with a tumor volume in the range of 80mm 3 to 150mm 3 and divide them into groups according to the average tumor volume (5 to 6 mice in each group) , administered on days 6/5, 9/8, 12/11, 15/14, 18/17, and 21/20 after inoculation, negative control antibody, positive control antibody (Taizhou Baiying Biotechnology Co., Ltd., Cat. No. B218501) Antigen-binding protein of the present application, monitor the changes in tumor volume and body weight of mice in each group during the administration period, the monitoring frequency is 2 times/week, and the monitoring is continuous for 3 weeks. Body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 25th/23rd day after inoculation, and the calculation formula was as follows: TGI (%)=[1-(T i -T 0 )/( V i -V 0 )]×100; where T i : mean tumor volume of drug-administered group, T 0 : mean tumor volume of drug-treated group on D0 day, Vi: mean tumor volume of same-type control group, V0: same-type control Group mean tumor volume on day D0. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: T V =1/2×a×b 2 . Body weight was measured using an electronic balance. The dosage and method of administration are shown in Table 10.
表10 给药试验设计Table 10 Drug administration test design
编号serial number 给药剂量Dosage 给药次数Dosing times 给药方式Method of administration
阴性对照抗体negative control antibody 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab0Ab0 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab1Ab1 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab2Ab2 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab3Ab3 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab4Ab4 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab5Ab5 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
Ab10Ab10 10mg/kg10mg/kg Q3DX6*Q3DX6* 腹腔注射intraperitoneal injection
*表示：每隔3天给药，共6次。*Indicates: Dosing every 3 days for a total of 6 doses.
第一次试验中抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4在人Siglec15转基因小鼠体内的抗肿瘤药效检测实验结果如图7A所示。第一次试验中抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4施用后，人Siglec15转基因小鼠的体重变化实验结果如图7B所示。第二次试验中抗原结合蛋白Ab0、Ab5和Ab10在人Siglec15转基因小鼠体内的抗肿瘤药效检测实验结果如图8A所示。第二次试验中抗原结合蛋白Ab0、Ab5和Ab10施用后，人Siglec15转基因小鼠的体重变化实验结果如图8B所示。The results of the anti-tumor efficacy test of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 in the human Siglec15 transgenic mice in the first experiment are shown in Figure 7A. After administration of the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 in the first experiment, the results of the body weight change experiment of the human Siglec15 transgenic mice are shown in Fig. 7B. The results of the anti-tumor efficacy test of the antigen-binding proteins Ab0, Ab5 and Ab10 in the human Siglec15 transgenic mice in the second experiment are shown in FIG. 8A . After administration of the antigen-binding proteins Ab0, Ab5 and Ab10 in the second experiment, the results of the body weight change experiment of the human Siglec15 transgenic mice are shown in FIG. 8B .
实验结果如表11和表12所示，第一次试验中在接种后第25天，本申请的抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4在10mg/kg剂量下显著性抑制肿瘤生长，肿瘤体积抑制率分别为87％、60％、51％、75％和83％。第二次试验中在接种后第23天，本申请的抗原结合蛋白Ab0、Ab5和Ab10在10mg/kg剂量下显著性抑制肿瘤的生长，肿瘤体积抑制率分别为55％、70％和32％。The experimental results are shown in Table 11 and Table 12. In the first test, on the 25th day after inoculation, the antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application significantly inhibited tumor growth at a dose of 10 mg/kg. The tumor volume inhibition rates were 87%, 60%, 51%, 75% and 83%, respectively. In the second test, on the 23rd day after inoculation, the antigen-binding proteins Ab0, Ab5 and Ab10 of the present application significantly inhibited the growth of tumors at a dose of 10 mg/kg, and the tumor volume inhibition rates were 55%, 70% and 32% respectively .
上述试验表明，本申请的抗原结合蛋白Ab0、Ab1、Ab2、Ab3、Ab4、Ab5和Ab10显著性抑制肿瘤的生长，并且对人Siglec15转基因小鼠体重均没有明显影响。The above experiments show that the antigen-binding proteins Ab0, Ab1, Ab2, Ab3, Ab4, Ab5 and Ab10 of the present application significantly inhibit the growth of tumors, and have no significant effect on the body weight of human Siglec15 transgenic mice.
表11 第一次试验中第25天本申请的抗原结合蛋白Ab0、Ab1、Ab2、Ab3和Ab4体内抑制肿瘤生长能力Table 11 Antigen-binding proteins Ab0, Ab1, Ab2, Ab3 and Ab4 of the present application have the ability to inhibit tumor growth in vivo on the 25th day in the first test
Figure PCTCN2022115816-appb-000012
Figure PCTCN2022115816-appb-000012
*表示：与阴性对照抗体肿瘤体积相比，P<0.05；**表示：与阴性对照抗体肿瘤体积相比，P<0.01。* indicates: compared with the negative control antibody tumor volume, P<0.05; ** indicates: compared with the negative control antibody tumor volume, P<0.01.
表12 第二次试验中第23天本申请的抗原结合蛋白Ab0、Ab5和Ab10体内抑制肿瘤生长能力Table 12 Antigen-binding proteins Ab0, Ab5 and Ab10 of the present application have the ability to inhibit tumor growth in vivo on the 23rd day in the second test
Figure PCTCN2022115816-appb-000013
Figure PCTCN2022115816-appb-000013
*表示：与阴性对照抗体肿瘤体积相比，P<0.05；**表示：与阴性对照抗体肿瘤体积相 比，P<0.01。* means: compared with the negative control antibody tumor volume, P<0.05; ** means: compared with the negative control antibody tumor volume, P<0.01.
实施例14 人源化Siglec15抗原结合蛋白抑制破骨细胞形成的活性Example 14 Humanized Siglec15 antigen-binding protein inhibits the activity of osteoclast formation
用TRAP Staining kit(来源：Cosmo Bio Co.,LTD，货号：AK04F)检测不同Siglec15抗原结合蛋白抑制破骨细胞形成的活性。The activity of different Siglec15 antigen-binding proteins in inhibiting osteoclast formation was detected with TRAP Staining kit (source: Cosmo Bio Co., LTD, product number: AK04F).
14.1将冻存的人外周单个核细胞(PBMC)在37°水浴锅迅速解冻，用Pan Monocyte Isolation Kit(来源：Miltenyi，货号：130-096-537)分选单核细胞。14.1 The frozen human peripheral mononuclear cells (PBMC) were thawed quickly in a 37° water bath, and the mononuclear cells were sorted with Pan Monocyte Isolation Kit (source: Miltenyi, catalog number: 130-096-537).
14.2离心计数，配制含25ng/mlM-SC(来源：peprotech，货号：AF-300-25)与30ng/mlRANKL(来源：peprotech，货号：AF-310-01)的带血清1640全培养基，调整细胞密度，铺于96孔板。14.2 Count by centrifugation, prepare serum-containing 1640 full medium containing 25ng/ml M-SC (source: peprotech, product number: AF-300-25) and 30ng/ml RANKL (source: peprotech, product number: AF-310-01), adjust Cell density, plated in 96-well plate.
14.3每孔加入100ul全培养基配制的抗体，终浓度为50nM。设置No-RANKL的培养基和全培养基(含M-CSF+RANKL)作为对照组。14.3 Add 100ul of antibody prepared in full medium to each well, with a final concentration of 50nM. No-RANKL medium and full medium (containing M-CSF+RANKL) were set as control groups.
14.4隔两天吸掉原来培养基，加入新鲜培养基和抗体,继续在培养箱培养。14.4 Aspirate the original medium every two days, add fresh medium and antibody, and continue culturing in the incubator.
14.5第8天收集上清，用50ulTRAP Staining Kit buffer与10ul上清共同孵育2h,在540nm波长处检测破骨分泌的抗酒石酸酸性磷酸酶的水平。14.5 Collect the supernatant on the 8th day, incubate with 50ulTRAP Staining Kit buffer and 10ul supernatant for 2h, and detect the level of tartrate-resistant acid phosphatase secreted by osteoclasts at a wavelength of 540nm.
以上试验表明，本申请人源化Siglec15抗原结合蛋白能够抑制破骨的形成。The above experiments show that the humanized Siglec15 antigen-binding protein of the present application can inhibit the formation of osteoclasts.
如图9所示，人源化Siglec15抗原结合蛋白能够抑制破骨细胞的形成，并且活性与阳性抗体相当。As shown in Figure 9, the humanized Siglec15 antigen-binding protein can inhibit the formation of osteoclasts, and the activity is comparable to that of the positive antibody.
实施例15 Siglec15抗原结合蛋白与PD-1抗体联用对人PD-1/PD-L1转基因小鼠的抗肿瘤药效试验Example 15 Antitumor efficacy test of Siglec15 antigen-binding protein combined with PD-1 antibody on human PD-1/PD-L1 transgenic mice
本试验采用MC38/hSiglec15细胞接种人PD-1/PD-L1转基因小鼠测定本发明的Siglec15抗原结合蛋白与PD-1抗体联用的抗肿瘤作用。In this experiment, MC38/hSiglec15 cells were used to inoculate human PD-1/PD-L1 transgenic mice to determine the anti-tumor effect of Siglec15 antigen-binding protein of the present invention combined with PD-1 antibody.
人PD-1/PD-L1转基因小鼠：雌性C57BL/6背景的人PD-1/PD-L1转基因小鼠(6周)购自上百奥赛图江苏基因生物技术有限公司。小鼠在到达后适应性饲养7天，随后开始研究。Human PD-1/PD-L1 transgenic mice: Female human PD-1/PD-L1 transgenic mice (6 weeks old) with C57BL/6 background were purchased from Shangbai Ocyto Jiangsu Gene Biotechnology Co., Ltd. Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
细胞：鼠源MC38细胞购自和元生物技术(上海)股份有限公司，按照说明书进行常规传代培养；对MC38细胞进行基因改造，使其过量表达人源Siglec15，该细胞被命名为MC38/人Siglec15细胞，用于后续体内实验。离心收集细胞，在无血清培养基中重悬细胞并调整细胞密度，在第0天将细胞悬液皮下接种至人PD-1/PD-L1转基因小鼠右腋窝皮下来建立MC38/人Siglec15荷瘤小鼠模型。Cells: Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and conventionally subcultured according to the instructions; MC38 cells were genetically modified to overexpress human Siglec15, and the cells were named MC38/human Siglec15 cells for subsequent in vivo experiments. Cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of human PD-1/PD-L1 transgenic mice to establish MC38/human Siglec15 loading. Tumor mouse model.
给药：Administration:
肿瘤细胞接种第7天，检测各只小鼠瘤体积，挑选出瘤体积在100mm 3～235mm 3范围内的小鼠按瘤体积平均分组(每组6只小鼠)，肿瘤细胞接种第7天单次给药，PD-1抗体(帕博利珠单抗Pembrolizumab，来源：泰州市百英生物科技有限公司，货号：B2014-CHO)；分别在接种后第7、10、14、17、21、24天给药，阴性对照抗体(人IgG)、阳性对照抗体(SPH-Sg-PC-1，来源：杭州皓阳生物技术有限公司，货号HSP067-41)、本申请抗原结合蛋白，给药期间监测各组小鼠瘤体积和体重变化，监测频率均为2次/周，连续监测3周。在每次给药前测定体重和肿瘤体积，接种后第28天计算肿瘤体积抑制率(TGI％)，计算公式如下：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100；其中Ti：给药组肿瘤体积均数，T0：给药组D0天的肿瘤体积均数，Vi：同型对照组肿瘤体积均数，V0：同型对照组D0天的肿瘤体积均数。肿瘤体积测定：采用游标卡尺测定肿瘤的长径(a)和宽径(b)，肿瘤体积按如下公式计算：TV＝1/2×a×b 2。采用电子天平测定体重。 On the 7th day after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with a tumor volume in the range of 100 mm 3 to 235 mm 3 were selected and grouped according to the average tumor volume (6 mice in each group). On the 7th day of tumor cell inoculation, Single administration, PD-1 antibody (pembrolizumab, source: Taizhou Baiying Biotechnology Co., Ltd., product number: B2014-CHO); 24 days of administration, negative control antibody (human IgG), positive control antibody (SPH-Sg-PC-1, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number HSP067-41), the antigen binding protein of this application, during the administration period The changes in tumor volume and body weight of mice in each group were monitored at a frequency of 2 times/week for 3 consecutive weeks. Body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 28th day after inoculation, and the calculation formula was as follows: TGI (%)=[1-(Ti-T0)/(Vi-V0) ]×100; where Ti: average tumor volume of the administration group, T0: average tumor volume of the administration group on D0 day, Vi: average tumor volume of the same-type control group, V0: average tumor volume of the same-type control group on D0 day . Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: TV=1/2×a×b 2 . Body weight was measured using an electronic balance.
给药剂量和方式如表13所示。The dosage and method of administration are shown in Table 13.
表13 给药试验设计Table 13 Dosing trial design
Figure PCTCN2022115816-appb-000014
Figure PCTCN2022115816-appb-000014
*每周2次给药，共6次。*Administered 2 times a week for a total of 6 times.
如图10A所示，抗原结合蛋白Ab5、Ab10与PD-1抗体联用在人PD-1/PD-L1转基因小鼠体内的抗肿瘤药效检测。As shown in Figure 10A, the anti-tumor drug efficacy of antigen-binding proteins Ab5 and Ab10 combined with PD-1 antibody in human PD-1/PD-L1 transgenic mice was tested.
如图10B所示，抗原结合蛋白Ab5、Ab10与PD-1抗体联用后，人PD-1/PD-L1转基因小鼠的体重变化。As shown in Figure 10B, the body weight of human PD-1/PD-L1 transgenic mice changed after antigen-binding proteins Ab5 and Ab10 were combined with PD-1 antibody.
如表13所示，在接种后第27天，本申请的抗原结合蛋白Ab5和Ab10在10mg/kg剂量下肿瘤体积抑制率分别为-1％和11％，PD-1抗体在1mg/kg剂量下肿瘤体积抑制率为9％； Ab5和PD-1抗体联用剂量下肿瘤体积抑制率为67％；Ab10和PD-1抗体联用剂量下肿瘤体积抑制率为63％；本申请的抗原结合蛋白Ab5和Ab10分别和PD-1抗体联用均显著性抑制肿瘤的生长。As shown in Table 13, on the 27th day after inoculation, the tumor volume inhibition rates of the antigen-binding proteins Ab5 and Ab10 of the present application at a dose of 10 mg/kg were -1% and 11%, respectively, and the PD-1 antibody at a dose of 1 mg/kg The tumor volume inhibition rate was 9%; the tumor volume inhibition rate was 67% under the combined dose of Ab5 and PD-1 antibody; the tumor volume inhibition rate was 63% under the combined dose of Ab10 and PD-1 antibody; The combination of protein Ab5 and Ab10 with PD-1 antibody can significantly inhibit the growth of tumor.
上述试验表明，本申请的抗原结合蛋白Ab5和Ab10分别和PD-1抗体联用均显著性抑制肿瘤的生长，并且对人PD-1/PD-L1转基因小鼠体重均没有明显影响。The above experiments showed that the combination of the antigen-binding proteins Ab5 and Ab10 of the present application with the PD-1 antibody significantly inhibited the growth of tumors, and had no significant effect on the body weight of human PD-1/PD-L1 transgenic mice.
表14 第28天本申请的抗原结合蛋白Ab5、Ab10和PD-1抗体联用的体内抑制肿瘤生长能力Table 14 Antigen-binding protein Ab5, Ab10 and PD-1 antibody combination of the present application on the 28th day inhibits tumor growth in vivo
Figure PCTCN2022115816-appb-000015
Figure PCTCN2022115816-appb-000015
*：与阴性对照抗体肿瘤体积相比，P<0.05。*: P<0.05 compared with negative control antibody tumor volume.
实施例16 Siglec15抗原结合蛋白与PD-L1抗体联用对人PD-1/PD-L1转基因小鼠的抗肿瘤药效试验Example 16 Antitumor efficacy test of Siglec15 antigen binding protein combined with PD-L1 antibody on human PD-1/PD-L1 transgenic mice
本试验采用MC38/hSiglec15细胞接种人PD-1/PD-L1转基因小鼠测定本发明的Siglec15抗原结合蛋白与PD-L1抗体联用的抗肿瘤作用。In this experiment, MC38/hSiglec15 cells were used to inoculate human PD-1/PD-L1 transgenic mice to determine the anti-tumor effect of Siglec15 antigen-binding protein of the present invention combined with PD-L1 antibody.
人PD-1/PD-L1转基因小鼠：雌性C57BL/6背景的人PD-1/PD-L1转基因小鼠(6周)购自上百奥塞图江苏基因生物技术有限公司。小鼠在到达后适应性饲养7天，随后开始研究。Human PD-1/PD-L1 transgenic mice: Female human PD-1/PD-L1 transgenic mice (6 weeks old) with C57BL/6 background were purchased from Shangbai Osset Jiangsu Gene Biotechnology Co., Ltd. Mice were acclimatized for 7 days upon arrival prior to initiation of the study.
细胞：鼠源MC38细胞购自和元生物技术(上海)股份有限公司，按照说明书进行常规传代培养；对MC38细胞进行基因改造，使其过量表达人源Siglec15，该细胞被命名为MC38/人Siglec15细胞，用于后续体内实验。离心收集细胞，在无血清培养基中重悬细胞并调整细胞密度，在第0天将细胞悬液皮下接种至人PD-1/PD-L1转基因小鼠右腋窝皮下来建立MC38/人Siglec15荷瘤小鼠模型。Cells: Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and conventionally subcultured according to the instructions; MC38 cells were genetically modified to overexpress human Siglec15, and the cells were named MC38/human Siglec15 cells for subsequent in vivo experiments. Cells were collected by centrifugation, and the cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of human PD-1/PD-L1 transgenic mice to establish MC38/human Siglec15 loading. Tumor mouse model.
给药：Administration:
肿瘤细胞接种第6天，检测各只小鼠瘤体积，挑选出瘤体积在120mm 3～160mm 3范围内的小鼠按瘤体积平均分组(每组6只小鼠)，分别在接种后第6、10、13、17、20天给药，阴性对照抗体(人IgG)、PD-L1抗体(阿特珠单抗Atezolizumab，来源：杭州皓阳生物技术有限公司，货号：HSP099-20)、阳性对照抗体(SPH-Sg-PC-1，来源：杭州皓阳生物技术有限公司，货号HSP067-41)、本申请抗原结合蛋白，给药期间监测各组小鼠瘤体积和体重变化，监测频率均为2次/周，连续监测3周。在每次给药前测定体重和肿瘤体积，接种后第24天计算肿瘤体积抑制率(TGI％)，计算公式如下：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100；其中Ti：给药组肿瘤体积均数，T0：给药组D0天的肿瘤体积均数，Vi：同型对照组肿瘤体积均数，V0：同型对照组D0天的肿瘤体积均数。肿瘤体积测定：采用游标卡尺测定肿瘤的长径(a)和宽径(b)，肿瘤体积按如下公式计算：TV＝1/2×a×b 2。采用电子天平测定体重。 On the 6th day after tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with a tumor volume in the range of 120 mm 3 to 160 mm 3 were selected and divided into groups according to the tumor volume (6 mice in each group). , 10, 13, 17, and 20 days of administration, negative control antibody (human IgG), PD-L1 antibody (atezolizumab, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number: HSP099-20), positive The control antibody (SPH-Sg-PC-1, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number HSP067-41), the antigen-binding protein of the application, and the changes in tumor volume and body weight of mice in each group were monitored during the administration, and the monitoring frequency was uniform. 2 times/week, continuous monitoring for 3 weeks. Body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 24th day after inoculation, and the calculation formula was as follows: TGI (%)=[1-(Ti-T0)/(Vi-V0) ]×100; where Ti: average tumor volume of the administration group, T0: average tumor volume of the administration group on D0 day, Vi: average tumor volume of the same-type control group, V0: average tumor volume of the same-type control group on D0 day . Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: TV=1/2×a×b 2 . Body weight was measured using an electronic balance.
给药剂量和方式如表15所示。The dosage and method of administration are shown in Table 15.
表15 给药试验设计Table 15 Drug administration trial design
编号serial number 给药剂量Dosage 给药次数Dosing times 给药方式Method of administration
阴性对照抗体negative control antibody 10mg/kg10mg/kg BIWX5*BIWX5* 腹腔注射intraperitoneal injection
PD-L1抗体PD-L1 antibody 3mg/kg3mg/kg BIWX5*BIWX5* 腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody 10mg/kg10mg/kg BIWX5*BIWX5* 腹腔注射intraperitoneal injection
Ab5Ab5 10mg/kg10mg/kg BIWX5*BIWX5* 腹腔注射intraperitoneal injection
Ab10Ab10 10mg/kg10mg/kg BIWX5*BIWX5* 腹腔注射intraperitoneal injection
PD-L1抗体+阳性对照抗体PD-L1 antibody + positive control antibody 3mg/kg+10mg/kg3mg/kg+10mg/kg BIWX5+BIWX5*BIWX5+BIWX5* 腹腔注射intraperitoneal injection
PD-L1抗体+Ab5PD-L1 antibody+Ab5 3mg/kg+10mg/kg3mg/kg+10mg/kg BIWX5+BIWX5*BIWX5+BIWX5* 腹腔注射intraperitoneal injection
PD-L1抗体+Ab10PD-L1 antibody+Ab10 3mg/kg+10mg/kg3mg/kg+10mg/kg BIWX5+BIWX5*BIWX5+BIWX5* 腹腔注射intraperitoneal injection
*每周2次给药，共5次。*Two doses per week for a total of 5 doses.
如图11A所示，抗原结合蛋白Ab5、Ab10与PD-L1抗体联用在人PD-1/PD-L1转基因小鼠体内的抗肿瘤药效检测。As shown in Figure 11A, the anti-tumor efficacy of antigen-binding proteins Ab5 and Ab10 combined with PD-L1 antibody in human PD-1/PD-L1 transgenic mice was tested.
如图11B所示，抗原结合蛋白Ab5、Ab10与PD-L1抗体联用后，人PD-1/PD-L1转基因小鼠的体重变化。As shown in Figure 11B, the body weight of human PD-1/PD-L1 transgenic mice changed after antigen-binding proteins Ab5 and Ab10 were combined with PD-L1 antibody.
如表16所示，在接种后第24天，本申请的抗原结合蛋白Ab5和Ab10在10mg/kg剂量下肿瘤体积抑制率分别为91％和63％，PD-L1抗体在3mg/kg剂量下肿瘤体积抑制率为58％；Ab5和PD-L1抗体联用剂量下肿瘤体积抑制率为101％；Ab10和PD-L1抗体联用剂量下肿瘤体积抑制率为98％；本申请的抗原结合蛋白Ab5和Ab10分别和PD-L1抗体联用均显著性抑 制肿瘤的生长。As shown in Table 16, on the 24th day after inoculation, the tumor volume inhibition rates of the antigen-binding proteins Ab5 and Ab10 of the present application at a dose of 10 mg/kg were 91% and 63%, respectively, and the PD-L1 antibody at a dose of 3 mg/kg The tumor volume inhibition rate was 58%; the tumor volume inhibition rate was 101% under the combined dose of Ab5 and PD-L1 antibody; the tumor volume inhibition rate was 98% under the combined dose of Ab10 and PD-L1 antibody; the antigen binding protein of the application Both Ab5 and Ab10 combined with PD-L1 antibody significantly inhibited tumor growth.
上述试验表明，本申请的抗原结合蛋白Ab5和Ab10分别和PD-L1抗体联用均显著性抑制肿瘤的生长，并且对人PD-1/PD-L1转基因小鼠体重均没有明显影响。The above experiments showed that the combination of the antigen-binding proteins Ab5 and Ab10 of the present application with the PD-L1 antibody significantly inhibited the growth of tumors, and had no significant effect on the body weight of human PD-1/PD-L1 transgenic mice.
表16 第24天本申请的抗原结合蛋白Ab5、Ab10和PD-L1抗体联用的体内抑制肿瘤生长能力Table 16 Antigen-binding protein Ab5, Ab10 and PD-L1 antibody combination of the present application on the 24th day inhibits tumor growth in vivo
Figure PCTCN2022115816-appb-000016
Figure PCTCN2022115816-appb-000016
**：与IgG对照抗体肿瘤体积相比，P<0.01。**: P<0.01 compared to IgG control antibody tumor volume.
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (92)

  1. 分离的抗原结合蛋白，其具有下述性质中的一种或多种：An isolated antigen binding protein having one or more of the following properties:
    a)在Biacore检测中，以约5E-09M或以下的K D值与人Siglec15蛋白特异性结合； a) in Biacore detection, specifically bind to human Siglec15 protein with a KD value of about 5E-09M or below;
    b)在流式检测中，以约0.2μg/ml或以下的EC 50值与表达于CHOK1细胞表面的人Siglec15特异性结合； b) specifically bind to human Siglec15 expressed on the surface of CHOK1 cells with an EC 50 value of about 0.2 μg/ml or less in flow cytometry;
    c)解除Siglec15对血液中PBMC的增殖抑制；c) releasing Siglec15 from inhibiting the proliferation of PBMCs in blood;
    d)在ELISA检测中，以约0.2μg/ml或以下的EC 50值与人Siglec15特异性结合； d) specifically bind to human Siglec15 with an EC 50 value of about 0.2 μg/ml or less in ELISA detection;
    e)在ELISA检测中，以约0.1μg/ml或以下的EC 50值与猴Siglec15特异性结合； e) specifically bind to monkey Siglec15 with an EC 50 value of about 0.1 μg/ml or below in ELISA detection;
    f)在ELISA检测中，以约0.1μg/ml或以下的EC 50值与鼠Siglec15特异性结合；以及 f) in an ELISA assay, specifically binds to mouse Siglec15 with an EC50 value of about 0.1 μg/ml or less; and
    g)抑制肿瘤细胞的生长和/或增殖。g) inhibiting the growth and/or proliferation of tumor cells.
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。The antigen binding protein of separation according to claim 1, it comprises HCDR3, and described HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:1.
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-2, which comprises HCDR2, said HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S。 The isolated antigen-binding protein according to any one of claims 1-3, which comprises HCDR1 comprising [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ]NYA wherein, X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, and X5 is S.
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:3或SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-4, which comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:20 or SEQ ID NO:22.
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。 The isolated antigen binding protein according to any one of claims 1-5, which comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ] the amino acid sequence shown in NYA, wherein, X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, X 5 is S, and described HCDR2 comprises SEQ ID NO:2 The amino acid sequence shown, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-6, which comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO:3, SEQ ID NO:20 or shown in SEQ ID NO:22 The amino acid sequence of the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
  8. 根据权利要求4-7中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:29所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 4-7, which comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H- FR1 comprises the amino acid sequence shown in SEQ ID NO:29.
  9. 根据权利要求8所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:9或SEQ ID NO:9所示的氨基酸序列。The isolated antigen-binding protein according to claim 8, wherein said H-FR1 comprises SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO: 9.
  10. 根据权利要求4-9中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:32所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 4-9, comprising H-FR2, said H-FR2 being located between said HCDR1 and said HCDR2, and said H-FR2 comprising SEQ ID The amino acid sequence shown in NO:32.
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:5或SEQ ID NO:10所示的氨基酸序列。The isolated antigen-binding protein according to claim 10, wherein the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10.
  12. 根据权利要求3-11中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:31所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 3-11, which comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises SEQ ID The amino acid sequence shown in NO:31.
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18或SEQ ID NO:27所示的氨基酸序列。The isolated antigen binding protein of claim 12, wherein said H-FR3 comprises SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:18 or SEQ ID NO:27 The amino acid sequence shown.
  14. 根据权利要求2-13中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 2-13, which comprises H-FR4, the N-terminus of said H-FR4 is directly or indirectly linked to the C-terminus of said HCDR3, and said H- FR4 comprises the amino acid sequence shown in SEQ ID NO:33.
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。The isolated antigen-binding protein according to claim 14, wherein the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:29所示的氨基酸序列，所述H-FR2包含SEQ ID NO:32所示的氨基酸序列，所述H-FR3包含SEQ ID NO:31所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-15, comprising H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NO:29 The amino acid sequence of the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:32, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:33 Amino acid sequence shown.
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10或SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14、SEQ ID NO:18或SEQ ID NO:27所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 1-16, which comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NO:4 or SEQ The amino acid sequence shown in ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:5, and the H-FR3 comprises SEQ ID NO:6, SEQ ID NO:11 , SEQ ID NO:14, SEQ ID NO:18 or the amino acid sequence shown in SEQ ID NO:27, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含选自下述任一组H-FR1，H-FR2，H-FR3和H-FR4；The isolated antigen-binding protein according to any one of claims 1-17, comprising H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups;
    1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；1) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
    2)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10 所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    3)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；3) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    4)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    5)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；5) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    6)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；6) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    7)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；7) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    8)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:27所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列。8) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:27 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12.
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:34所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 1-18, which comprises a heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO:34.
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:28所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 1-19, which comprises a heavy chain variable region VH comprising SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID The amino acid sequence shown in NO:28.
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含重链恒定区，且所述重链恒定区包含源自IgG的恒定区。The isolated antigen binding protein according to any one of claims 1-20, which comprises a heavy chain constant region, and said heavy chain constant region comprises an IgG-derived constant region.
  22. 根据权利要求21所述的分离的抗原结合蛋白，其中所述重链恒定区包括源自人IgG的恒定区。The isolated antigen binding protein of claim 21, wherein the heavy chain constant region comprises a constant region derived from human IgG.
  23. 根据权利要求21-22中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含 SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37或SEQ ID NO:38所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 21-22, wherein the heavy chain constant region comprises SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38 Amino acid sequence shown.
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包括抗体或抗原结合片段。The isolated antigen binding protein of any one of claims 1-23, which comprises an antibody or antigen binding fragment.
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述抗原结合片段选自：Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv，VHH和/或dAb。 The isolated antigen-binding protein of claim 24, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  26. 根据权利要求24-25中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。The isolated antigen binding protein according to any one of claims 24-25, wherein said antibody is selected from the group consisting of monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.
  27. 根据权利要求1-26中任一项所述的分离的抗原结合蛋白，其为单域抗体。The isolated antigen binding protein of any one of claims 1-26, which is a single domain antibody.
  28. 根据权利要求27所述的分离的抗原结合蛋白，其中所述单域抗体包含HCDR3，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。The isolated antigen-binding protein according to claim 27, wherein the single domain antibody comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1.
  29. 根据权利要求27-28中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含HCDR2，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 27-28, wherein said single domain antibody comprises HCDR2, said HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.
  30. 根据权利要求27-29中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含HCDR1，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S。 The isolated antigen binding protein according to any one of claims 27-29, wherein said single domain antibody comprises HCDR1 comprising [X 1 ][X 2 ][X 3 ][X 4 ][X 5 ] The amino acid sequence shown in NYA, wherein X1 is G or R, X2 is F or S, X3 is I or T, X4 is F, and X5 is S.
  31. 根据权利要求30中所述的分离的抗原结合蛋白，其中所述单域抗体的所述HCDR1包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列。The isolated antigen-binding protein according to claim 30, wherein said HCDR1 of said single domain antibody comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:20 or SEQ ID NO:22.
  32. 根据权利要求27-31中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含HCDR1，HCDR2和HCDR3，所述HCDR1包含[X 1][X 2][X 3][X 4][X 5]NYA所示的氨基酸序列，其中，X 1为G或R，X 2为F或S，X 3为I或T，X 4为F，X 5为S，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。 The isolated antigen binding protein according to any one of claims 27-31, wherein said single domain antibody comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising [X 1 ][X 2 ][X 3 ][X 4 ] The amino acid sequence shown in [X 5 ]NYA, wherein X 1 is G or R, X 2 is F or S, X 3 is I or T, X 4 is F, X 5 is S, and the HCDR2 comprises The amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1.
  33. 根据权利要求27-32中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3、SEQ ID NO:20或SEQ ID NO:22所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 27-32, wherein said single domain antibody comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO:3, SEQ ID NO:20 or SEQ ID The amino acid sequence shown in NO:22, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:1.
  34. 根据权利要求30-33中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:29所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 30-33, wherein the single domain antibody comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, And the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:29.
  35. 根据权利要求34所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR1，其中所述H-FR1包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列。The isolated antigen-binding protein of claim 34, wherein the single domain antibody comprises H-FR1, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:9.
  36. 根据权利要求30-35中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR2，所述H-FR2位于所述HCDR1和所述HCDR2之间，且所述H-FR2包含SEQ ID NO:32所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 30-35, wherein said single domain antibody comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H -FR2 comprises the amino acid sequence shown in SEQ ID NO:32.
  37. 根据权利要求36中所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR2，所述H-FR2包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列。The isolated antigen-binding protein according to claim 36, wherein the single domain antibody comprises H-FR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:9.
  38. 根据权利要求28-37中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR3，所述H-FR3位于所述HCDR2和所述HCDR3之间，且所述H-FR3包含SEQ ID NO:31所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 28-37, wherein said single domain antibody comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H -FR3 comprises the amino acid sequence shown in SEQ ID NO:31.
  39. 根据权利要求38中所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR3，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14或SEQ ID NO:18所示的氨基酸序列。The isolated antigen binding protein according to claim 38, wherein said single domain antibody comprises H-FR3, said H-FR3 comprising SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:14 or SEQ ID NO:11 The amino acid sequence shown in ID NO:18.
  40. 根据权利要求28-39中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 28-39, wherein the single domain antibody comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, And the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:33.
  41. 根据权利要求40中所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR4，所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。The isolated antigen-binding protein according to claim 40, wherein the single domain antibody comprises H-FR4, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  42. 根据权利要求27-41中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:29所示的氨基酸序列，所述H-FR2包含SEQ ID NO:32所示的氨基酸序列，所述H-FR3包含SEQ ID NO:31所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:33所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 27-41, wherein said single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID The amino acid sequence shown in NO:29, the H-FR2 includes the amino acid sequence shown in SEQ ID NO:32, the H-FR3 includes the amino acid sequence shown in SEQ ID NO:31, and the H-FR4 includes Amino acid sequence shown in SEQ ID NO:33.
  43. 根据权利要求27-42中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:4或SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5或SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:14或SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7或SEQ ID NO:12所示的氨基酸序列。The isolated antigen binding protein according to any one of claims 27-42, wherein said single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID The amino acid sequence shown in NO:4 or SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:10, and the H-FR3 comprises SEQ ID NO:6, The amino acid sequence shown in SEQ ID NO:11, SEQ ID NO:14 or SEQ ID NO:18, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:12.
  44. 根据权利要求27-43中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含选自下述任一组H-FR1，H-FR2，H-FR3和H-FR4；The isolated antigen-binding protein according to any one of claims 27-43, wherein the single domain antibody comprises H-FR1, H-FR2, H-FR3 and H-FR4 selected from any one of the following groups;
    1)所述H-FR1包含SEQ ID NO:4所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:7所示的氨基酸序列；1) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;
    2)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；2) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    3)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；3) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    4)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:11所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；4) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    5)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:14所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；5) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    6)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；6) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    7)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:18所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列；7) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 12;
    8)所述H-FR1包含SEQ ID NO:9所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5所示的氨基酸序列，所述H-FR3包含SEQ ID NO:27所示的氨基酸序列，且所述H-FR4包含SEQ ID NO:12所示的氨基酸序列。8) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:27 Amino acid sequence, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:12.
  45. 根据权利要求27-44中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含重链可变区VH，所述VH包含SEQ ID NO:34所示的氨基酸序列。The isolated antigen-binding protein according to any one of claims 27-44, wherein said single domain antibody comprises a heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO:34.
  46. 根据权利要求45中所述的分离的抗原结合蛋白，其中所述单域抗体包含重链可变区VH，所述VH包含SEQ ID NO:8、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:28所示的氨基酸序列。The isolated antigen binding protein according to claim 45, wherein said single domain antibody comprises a heavy chain variable region VH comprising SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID The amino acid sequence shown in NO:28.
  47. 根据权利要求27-46中任一项所述的分离的抗原结合蛋白，其中所述单域抗体包含重链恒定区，且所述重链恒定区包含源自IgG的恒定区。The isolated antigen binding protein of any one of claims 27-46, wherein the single domain antibody comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region.
  48. 根据权利要求47所述的分离的抗原结合蛋白，其中所述重链恒定区包括源自人IgG的恒定区。The isolated antigen binding protein of claim 47, wherein the heavy chain constant region comprises a constant region derived from human IgG.
  49. 根据权利要求47-48中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37或SEQ ID NO:38所示的氨基酸序列。The isolated antigen binding protein of any one of claims 47-48, wherein the heavy chain constant region comprises SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38 Amino acid sequence shown.
  50. 多肽，其包含权利要求1-49中任一项所述的分离的抗原结合蛋白。A polypeptide comprising the isolated antigen binding protein of any one of claims 1-49.
  51. 免疫缀合物，其包含权利要求1-49中任一项所述的分离的抗原结合蛋白或权利要求50所述的多肽。An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-49 or the polypeptide of claim 50.
  52. 分离的核酸分子，其编码权利要求1-49中任一项所述的分离的抗原结合蛋白，或者权利要求50所述的多肽。An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-49, or the polypeptide of claim 50.
  53. 载体，其包含权利要求52所述的分离的核酸分子。A vector comprising the isolated nucleic acid molecule of claim 52.
  54. 细胞，其包含权利要求1-49中任一项所述的分离的抗原结合蛋白，权利要求50所述的多肽，权利要求51所述的免疫缀合物，权利要求52所述的分离的核酸分子和/或权利要求53所述的载体。A cell comprising the isolated antigen binding protein of any one of claims 1-49, the polypeptide of claim 50, the immunoconjugate of claim 51, the isolated nucleic acid of claim 52 Molecule and/or the carrier described in claim 53.
  55. 制备权利要求1-49中任一项所述的分离的抗原结合蛋白或权利要求50所述的多肽的方法，所述方法包括再使得权利要求1-49中任一项所述的分离的抗原结合蛋白或权利要求50所述的多肽表达的条件下，培养权利要求54所述的细胞。A method for preparing the isolated antigen-binding protein of any one of claims 1-49 or the polypeptide of claim 50, said method comprising further making the isolated antigen of any one of claims 1-49 The cell according to claim 54 is cultured under the condition that the binding protein or the polypeptide according to claim 50 is expressed.
  56. 药物组合物，其包含权利要求1-49中任一项所述的分离的抗原结合蛋白，权利要求50所述的多肽，权利要求51所述的免疫缀合物，权利要求52所述的分离的核酸分子，权利要求53所述的载体，权利要求54所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-49, the polypeptide of claim 50, the immunoconjugate of claim 51, the isolated antigen binding protein of claim 52 The nucleic acid molecule of claim 53, the carrier of claim 54, the cell of claim 54, and/or pharmaceutically acceptable adjuvants and/or excipients.
  57. 药物组合，其包含权利要求1-49中任一项所述的分离的抗原结合蛋白和免疫检查点抑制剂。A pharmaceutical combination comprising the isolated antigen binding protein of any one of claims 1-49 and an immune checkpoint inhibitor.
  58. 根据权利要求57所述的药物组合，其中所述免疫检查点抑制剂包括抑制PD-1/PD-L1相互作用的物质。The drug combination according to claim 57, wherein the immune checkpoint inhibitor comprises a substance that inhibits PD-1/PD-L1 interaction.
  59. 根据权利要求57-58中任一项所述的药物组合，其中所述免疫检查点抑制剂选自下组：PD-1/PD-L1阻断剂、PD-1拮抗剂、PD-L1拮抗剂、PD-1抑制剂和PD-L1抑制剂。The drug combination according to any one of claims 57-58, wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1/PD-L1 blockers, PD-1 antagonists, PD-L1 antagonists agents, PD-1 inhibitors and PD-L1 inhibitors.
  60. 根据权利要求57-59中任一项所述的药物组合，其中所述免疫检查点抑制剂包括抗PD-1抗体。The pharmaceutical combination according to any one of claims 57-59, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody.
  61. 根据权利要求60所述的药物组合，其中所述抗PD-1抗体包含HCDR3，所述HCDR3包含SEQ ID NO:39所示的氨基酸序列。The pharmaceutical combination according to claim 60, wherein the anti-PD-1 antibody comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:39.
  62. 根据权利要求60-61中任一项所述的药物组合，其中所述抗PD-1抗体包含HCDR2，所述HCDR2包含SEQ ID NO:40所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-61, wherein the anti-PD-1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:40.
  63. 根据权利要求60-62中任一项所述的药物组合，其中所述抗PD-1抗体包含HCDR1，所 述HCDR1包含SEQ ID NO:41所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-62, wherein the anti-PD-1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:41.
  64. 根据权利要求60-63中任一项所述的药物组合，其中所述抗PD-1抗体包含重链可变区VH，所述VH包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:39所示的氨基酸序列；所述HCDR2包含SEQ ID NO:40所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:42所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-63, wherein the anti-PD-1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises SEQ ID NO: The amino acid sequence shown in 39; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:40; And the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:42.
  65. 根据权利要求60-64中任一项所述的药物组合，其中所述抗PD-1抗体包含重链可变区VH，所述VH包含SEQ ID NO:42所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-64, wherein the anti-PD-1 antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:42.
  66. 根据权利要求60-65中任一项所述的药物组合，其中所述抗PD-1抗体包含LCDR3，所述LCDR3包含SEQ ID NO:43所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-65, wherein the anti-PD-1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:43.
  67. 根据权利要求60-66中任一项所述的药物组合，其中所述抗PD-1抗体包含LCDR2，所述LCDR2包含SEQ ID NO:44所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-66, wherein the anti-PD-1 antibody comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:44.
  68. 根据权利要求60-67中任一项所述的药物组合，其中所述抗PD-1抗体包含LCDR1，所述LCDR1包含SEQ ID NO:45所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-67, wherein the anti-PD-1 antibody comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45.
  69. 根据权利要求60-68中任一项所述的药物组合，其中所述抗PD-1抗体包含轻链可变区VL，所述VL包含LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:43所示的氨基酸序列；所述LCDR2包含SEQ ID NO:44所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:45所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-68, wherein the anti-PD-1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises SEQ ID NO: The amino acid sequence shown in 43; The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:44; And the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:45.
  70. 根据权利要求60-69中任一项所述的药物组合，其中所述抗PD-1抗体包含轻链可变区VL，所述VL包含SEQ ID NO:46所示的氨基酸序列。The pharmaceutical combination according to any one of claims 60-69, wherein the anti-PD-1 antibody comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:46.
  71. 根据权利要求60-70中任一项所述的药物组合，其中所述抗PD-1抗体包括帕博丽珠单抗。The pharmaceutical combination according to any one of claims 60-70, wherein the anti-PD-1 antibody comprises pembrolizumab.
  72. 根据权利要求57-71中任一项所述的药物组合，其中所述免疫检查点抑制剂包括抗PD-L1抗体。The pharmaceutical combination according to any one of claims 57-71, wherein the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.
  73. 根据权利要求72所述的药物组合，其中所述抗PD-L1抗体包含HCDR3，所述HCDR3包含SEQ ID NO:47所示的氨基酸序列。The pharmaceutical combination according to claim 72, wherein the anti-PD-L1 antibody comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:47.
  74. 根据权利要求72-73中任一项所述的药物组合，其中所述PD-L1抗体包含HCDR2，所述HCDR2包含SEQ ID NO:48所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-73, wherein the PD-L1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:48.
  75. 根据权利要求72-74中任一项所述的药物组合，其中所述PD-L1抗体包含HCDR1，所述HCDR1包含SEQ ID NO:49所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-74, wherein the PD-L1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:49.
  76. 根据权利要求72-75中任一项所述的药物组合，其中所述PD-L1抗体包含重链可变区VH，所述VH包含HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:47所 示的氨基酸序列；所述HCDR2包含SEQ ID NO:48所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:49所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-75, wherein the PD-L1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR3 comprises SEQ ID NO:47 The amino acid sequence shown; the HCDR2 includes the amino acid sequence shown in SEQ ID NO:48; and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:49.
  77. 根据权利要求72-76中任一项所述的药物组合，其中所述抗PD-L1抗体包含重链可变区VH，所述VH包含SEQ ID NO:50所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-76, wherein the anti-PD-L1 antibody comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:50.
  78. 根据权利要求72-77中任一项所述的药物组合，其中所述抗PD-L1抗体包含LCDR3，所述LCDR3包含SEQ ID NO:51所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-77, wherein the anti-PD-L1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:51.
  79. 根据权利要求72-78中任一项所述的药物组合，其中所述抗PD-L1抗体包含LCDR2，所述LCDR2包含SEQ ID NO:52所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-78, wherein the anti-PD-L1 antibody comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:52.
  80. 根据权利要求72-79中任一项所述的药物组合，其中所述抗PD-L1抗体包含LCDR1，所述LCDR1包含SEQ ID NO:53所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-79, wherein the anti-PD-L1 antibody comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:53.
  81. 根据权利要求72-80中任一项所述的药物组合，其中所述抗PD-L1抗体包含轻链可变区VL，所述VL包含LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:51所示的氨基酸序列；所述LCDR2包含SEQ ID NO:53所示的氨基酸序列；以及所述LCDR1包含SEQ ID NO:53所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-80, wherein the anti-PD-L1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, and the LCDR3 comprises SEQ ID NO: The amino acid sequence shown in 51; The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:53; And the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:53.
  82. 根据权利要求72-81中任一项所述的药物组合，其中所述抗PD-L1抗体包含轻链可变区VL，所述VL包含SEQ ID NO:54所示的氨基酸序列。The pharmaceutical combination according to any one of claims 72-81, wherein the anti-PD-L1 antibody comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:54.
  83. 根据权利要求72-82中任一项所述的药物组合，其中所述抗PD-L1抗体包括阿替利珠单抗。The pharmaceutical combination according to any one of claims 72-82, wherein the anti-PD-L1 antibody comprises atezolizumab.
  84. 一种用于检测或测定Siglec15的方法，所述方法包括使用权利要求1-49中任一项所述的分离的抗原结合蛋白或权利要求50所述的多肽。A method for detecting or measuring Siglec15, said method comprising using the isolated antigen-binding protein of any one of claims 1-49 or the polypeptide of claim 50.
  85. 一种Siglec15的检测试剂盒，其包含权利要求1-49中任一项所述的分离的抗原结合蛋白或权利要求50所述的多肽。A detection kit for Siglec15, comprising the isolated antigen-binding protein of any one of claims 1-49 or the polypeptide of claim 50.
  86. 权利要求1-49中任一项所述的分离的抗原结合蛋白或权利要求50所述的多肽在制备试剂盒中的用途。Use of the isolated antigen-binding protein of any one of claims 1-49 or the polypeptide of claim 50 in the preparation of a kit.
  87. 一种调节免疫应答的方法，其包括向有需要的受试者施用有效量的权利要求1-49中任一项所述的分离的抗原结合蛋白，权利要求50所述的多肽，权利要求51所述的免疫缀合物，权利要求52所述的分离的核酸分子，权利要求53所述的载体，权利要求54所述的细胞，权利要求56所述的药物组合物，和/或权利要求57-83中任一项所述的药物组合，和/或药学上可接受的治疗剂。A method of modulating an immune response comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein of any one of claims 1-49, the polypeptide of claim 50, the polypeptide of claim 51 The immunoconjugate, the isolated nucleic acid molecule of claim 52, the carrier of claim 53, the cell of claim 54, the pharmaceutical composition of claim 56, and/or the vector of claim 53 The pharmaceutical combination described in any one of 57-83, and/or a pharmaceutically acceptable therapeutic agent.
  88. 权利要求1-49中任一项所述的分离的抗原结合蛋白，权利要求50所述的多肽，权利要求51所述的免疫缀合物，权利要求52所述的分离的核酸分子，权利要求53所述的载 体，权利要求54所述的细胞，权利要求56所述的药物组合物和/或权利要求57-83中任一项所述的药物组合在制备预防和/或治疗疾病或病症的药物中的用途。The isolated antigen binding protein of any one of claims 1-49, the polypeptide of claim 50, the immunoconjugate of claim 51, the isolated nucleic acid molecule of claim 52, the polypeptide of claim 52, The carrier described in 53, the cell described in claim 54, the pharmaceutical composition described in claim 56 and/or the pharmaceutical combination described in any one of claims 57-83 are prepared to prevent and/or treat diseases or illnesses use in medicines.
  89. 根据权利要求88所述的用途，其中所述疾病或病症包括肿瘤。The use according to claim 88, wherein the disease or condition comprises a tumor.
  90. 根据权利要求88-89中任一项所述的用途，其中所述肿瘤包括与Siglec15的表达相关的肿瘤。The use according to any one of claims 88-89, wherein the tumor comprises a tumor associated with the expression of Siglec15.
  91. 根据权利要求89-90中任一项所述的用途，其中所述肿瘤包括实体瘤。The use according to any one of claims 89-90, wherein the tumor comprises a solid tumor.
  92. 根据权利要求89-91中任一项所述的用途，其中所述肿瘤包括结肠癌。The use according to any one of claims 89-91, wherein the tumor comprises colon cancer.
PCT/CN2022/115816 2021-08-31 2022-08-30 Antigen-binding protein targeting siglec15 and use thereof WO2023030311A1 (en)

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