WO2023030311A1

WO2023030311A1 - Antigen-binding protein targeting siglec15 and use thereof

Info

Publication number: WO2023030311A1
Application number: PCT/CN2022/115816
Authority: WO
Inventors: 廖雪梅; 杨松霖; 陈娜; 王瓅; 施伟军; 栾珊珊; 夏广新; 柯樱
Original assignee: 上海医药集团股份有限公司
Priority date: 2021-08-31
Filing date: 2022-08-30
Publication date: 2023-03-09
Also published as: CN118043351A

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

Antigen-binding protein targeting Siglec15 and use thereof

technical field

This application relates to the field of biomedicine, in particular to an antigen-binding protein targeting Siglec15 and its use.

Background technique

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.

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.

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.

Contents of the invention

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.

In certain embodiments, the isolated antigen-binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:1.

In certain embodiments, the isolated antigen-binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 .

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.

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.

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.

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.

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.

In certain embodiments, said heavy chain constant region of said isolated antigen binding protein comprises a constant region derived from human IgG.

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 .

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment.

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.

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.

In certain embodiments, the isolated antigen binding protein is a single domain antibody.

In certain embodiments, the single domain antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:1.

In certain embodiments, the single domain antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2.

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.

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.

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.

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.

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.

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.

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 .

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.

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 .

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.

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.

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.

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.

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.

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.

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.

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 .

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.

In certain embodiments, said heavy chain constant region of said single domain antibody comprises a constant region derived from human IgG.

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.

In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein described herein.

In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein or the polypeptide described herein.

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.

In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described herein.

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.

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.

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.

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.

In certain embodiments, the immune checkpoint inhibitor includes a substance that inhibits PD-1/PD-L1 interaction.

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.

In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody.

In certain embodiments, the anti-PD-1 antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:39.

In certain embodiments, the anti-PD-1 antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:40.

In certain embodiments, the anti-PD-1 antibody comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:41.

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.

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.

In certain embodiments, the anti-PD-1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:43.

In certain embodiments, the anti-PD-1 antibody comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO:44.

In certain embodiments, the anti-PD-1 antibody comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:45.

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.

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.

In certain embodiments, the anti-PD-1 antibody comprises pembrolizumab.

In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.

In certain embodiments, the anti-PD-L1 antibody comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:47.

In certain embodiments, the anti-PD-L1 antibody comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:48.

In certain embodiments, the anti-PD-L1 antibody comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:49.

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.

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.

In certain embodiments, the anti-PD-L1 antibody comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:51.

In certain embodiments, the anti-PD-L1 antibody comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:52.

In certain embodiments, the anti-PD-L1 antibody comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:53.

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.

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.

In certain embodiments, the anti-PD-L1 antibody comprises atezolizumab.

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.

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.

In another aspect, please provide a use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit.

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.

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.

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.

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.

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.

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.

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.

Description of drawings

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:

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.

Figure 2A shows that the Siglec15 antigen-binding protein described in this application promotes the proliferation of CD8 ⁺ T cells.

Figure 2B shows that the Siglec15 antigen-binding protein described in this application promotes the proliferation of CD4 ⁺ T cells.

Figure 2C shows that the Siglec15 antigen-binding protein described in this application promotes the expression of IFN-γ 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.

Figure 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-γ 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.

Detailed ways

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.

Definition of Terms

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In this application, the term "and/or" should be understood as meaning any one of the optional items or both of the optional items.

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%.

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".

Detailed description of the invention

isolated antigen binding protein

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.

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.

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.

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.

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.

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.

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.

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.

In this application, the isolated antigen-binding protein may be a single domain antibody.

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.

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.

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.

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.

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.

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.

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.

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), 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.

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.

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.

For example, the VH of the antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3 and/or H-FR4.

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.

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), wherein X ₁ can be A or E.

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.

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.

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), wherein X ₁ can be G or Q, and X ₂ can be L or R.

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.

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.

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), 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.

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.

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.

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), wherein X ₁ can be M or Q.

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.

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.

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.

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) 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) 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) 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) 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) 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) 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) 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) 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.

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.

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.

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.

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.

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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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.

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), 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.

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).

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.

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.

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.

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.

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.

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.

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.

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), 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.

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.

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).

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 .

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).

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.

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.

Peptides and Immunoconjugates

In another aspect, the application provides one or more polypeptides, which may comprise the isolated antigen binding proteins of the application and fragments thereof.

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.

Nucleic acids, vectors, cells and pharmaceutical compositions

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. )

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).

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.

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.

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.

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.

drug combination

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.

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.

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.

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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).

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.

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.

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.

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).

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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).

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.

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.

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.

The drug combination described in this application can effectively kill tumors.

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.

Kits, methods, uses

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) contacting a sample with an antigen binding protein of the present application; and

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Example

Embodiment 1 immune alpaca

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.

Example 2 Construction of alpaca immune library

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.

Example 3 Screening of anti-human Siglec15 antigen-binding protein

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.

Example 4 Humanized Siglec15 antigen-binding protein

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.

Table 1 The variable region sequence information of the humanized antigen-binding protein

Example 5 Expression and purification of Siglec15 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 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 affinity chromatography column purification of antibody protein:

(1) Equilibrium chromatography column: 1×PBS, flow rate 1ml/min, 20ml

(2) Sample loading: flow rate 1ml/min

(3) Washing: 1×PBS, flow rate 1ml/min, 20ml

(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) 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.

Example 6 Binding of Siglec15 antigen-binding protein to human Siglec15 expressed on cells

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. Spread CHOK1/hSiglec15 cells (Kangyuan Bochuang) in a 96-well plate.

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. Wash the plate 3 times with PBS containing 2% FBS.

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. Wash the plate twice with PBS containing 2% FBS.

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.

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.

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

Example 7 Biological activity of Siglec15 antigen-binding protein

7.1 Detection of CD4 and CD8 T cell proliferation

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. 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. 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. 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. 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. 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 Use human IFN-g Elisa Kit (RnD, Cat. No. DY285B) to detect the expression of IFN-γ in the medium supernatant

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. Use PBST (0.05%

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. 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. 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. 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. 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. 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. 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.

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.

Table 3 The ability of Siglec15 binding to block Siglec15 to inhibit the proliferation of PBMC in the blood of subjects

Example 8 Binding of humanized Siglec15-binding protein to human Siglec15 expressed on cells

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. Spread CHOK1/hSiglec15 cells (Kangyuan Bochuang) in a 96-well plate.

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. Wash the plate 3 times with PBS containing 2% FBS.

5. Wash the plate twice with PBS containing 2% FBS.

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

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

Example 9 Biological activity of humanized Siglec15 binding protein

9.1 Detection of CD4 and CD8 T cell proliferation

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. 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. 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. 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. 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. 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 Use human IFN-g Elisa Kit (RnD, Cat. No. DY285B) to detect the expression of IFN-γ in the medium supernatant

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. Use PBST (0.05%

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. 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. 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. 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. 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. 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.

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.

Table 5 Siglec15 antigen-binding protein blocks the ability of Siglec15 to inhibit the proliferation of PBMC in the blood of subjects

Example 10 Binding affinity of humanized Siglec15 antigen-binding protein

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. 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. 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. 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. 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. 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.

The above tests show that the humanized Siglec15 antigen-binding protein of the present application has binding affinity with antigen.

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.

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

Example 11 Species cross-reactivity test of humanized Siglec15 antigen-binding protein

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. 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. Discard the coating solution the next day, and wash the plate 3 times with 0.05% Tween20 PBS.

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. 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. 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. Add 100ul TMB substrate chromogenic solution to each well and incubate at room temperature for 15 minutes.

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.

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

Example 12 Antitumor efficacy test of Siglec15 antigen-binding protein on wild-type C57BL/6N animal model

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 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.

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.

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.

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	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab0Ab0	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab1Ab1	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab2Ab2	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab3Ab3	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection

Ab4

10mg/kg

Q3DX6*

intraperitoneal injection

*Indicates: Dosing every 3 days for a total of 6 doses.

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.

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

** indicates: P<0.01 compared with the negative control antibody tumor volume.

Example 13 Antitumor efficacy test of Siglec15 antigen-binding protein on human Siglec15 transgenic mice

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.

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.

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.

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.

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	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab0Ab0	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab1Ab1	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab2Ab2	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab3Ab3	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab4Ab4	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab5Ab5	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab10Ab10	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection

*Indicates: Dosing every 3 days for a total of 6 doses.

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. 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 .

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.

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

* indicates: compared with the negative control antibody tumor volume, P<0.05; ** indicates: compared with the negative control antibody tumor volume, P<0.01.

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

* means: compared with the negative control antibody tumor volume, P<0.05; ** means: compared with the negative control antibody tumor volume, P<0.01.

Example 14 Humanized Siglec15 antigen-binding protein inhibits the activity of osteoclast formation

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 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 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 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 Aspirate the original medium every two days, add fresh medium and antibody, and continue culturing in the incubator.

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.

The above experiments show that the humanized Siglec15 antigen-binding protein of the present application can inhibit the formation of osteoclasts.

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.

Example 15 Antitumor efficacy test of Siglec15 antigen-binding protein combined with PD-1 antibody on human PD-1/PD-L1 transgenic mice

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.

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.

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.

Administration:

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.

The dosage and method of administration are shown in Table 13.

Table 13 Dosing trial design

*Administered 2 times a week for a total of 6 times.

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.

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.

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.

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.

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

*: P<0.05 compared with negative control antibody tumor volume.

Example 16 Antitumor efficacy test of Siglec15 antigen binding protein combined with PD-L1 antibody on human PD-1/PD-L1 transgenic mice

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.

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.

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 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.

The dosage and method of administration are shown in Table 15.

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	BIWX5BIWX5	腹腔注射intraperitoneal injection
PD-L1抗体PD-L1 antibody	3mg/kg3mg/kg	BIWX5BIWX5	腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody	10mg/kg10mg/kg	BIWX5BIWX5	腹腔注射intraperitoneal injection
Ab5Ab5	10mg/kg10mg/kg	BIWX5BIWX5	腹腔注射intraperitoneal injection
Ab10Ab10	10mg/kg10mg/kg	BIWX5BIWX5	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+BIWX5BIWX5+BIWX5	腹腔注射intraperitoneal injection
PD-L1抗体+Ab5PD-L1 antibody+Ab5	3mg/kg+10mg/kg3mg/kg+10mg/kg	BIWX5+BIWX5BIWX5+BIWX5	腹腔注射intraperitoneal injection
PD-L1抗体+Ab10PD-L1 antibody+Ab10	3mg/kg+10mg/kg3mg/kg+10mg/kg	BIWX5+BIWX5BIWX5+BIWX5	腹腔注射intraperitoneal injection

*Two doses per week for a total of 5 doses.

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.

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.

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.

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.

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

**: P<0.01 compared to IgG control antibody tumor volume.

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

An isolated antigen binding protein having one or more of the following properties:

a) in Biacore detection, specifically bind to human Siglec15 protein with a KD value of about 5E-09M or below;

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) releasing Siglec15 from inhibiting the proliferation of PBMCs in blood;

d) specifically bind to human Siglec15 with an EC 50 value of about 0.2 μg/ml or less in ELISA detection;

e) specifically bind to monkey Siglec15 with an EC 50 value of about 0.1 μg/ml or below in ELISA detection;

f) in an ELISA assay, specifically binds to mouse Siglec15 with an EC50 value of about 0.1 μg/ml or less; and

g) inhibiting the growth and/or proliferation of tumor cells.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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) 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) 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) 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) 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) 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) 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) 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) 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.
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.
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.
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.
The isolated antigen binding protein of claim 21, wherein the heavy chain constant region comprises a constant region derived from human IgG.
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.
The isolated antigen binding protein of any one of claims 1-23, which comprises an antibody or antigen binding fragment.
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.
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.
The isolated antigen binding protein of any one of claims 1-26, which is a single domain antibody.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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) 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) 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) 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) 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) 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) 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) 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) 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.
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.
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.
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.
The isolated antigen binding protein of claim 47, wherein the heavy chain constant region comprises a constant region derived from human IgG.
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.
A polypeptide comprising the isolated antigen binding protein of any one of claims 1-49.
An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-49 or the polypeptide of claim 50.
An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-49, or the polypeptide of claim 50.
A vector comprising the isolated nucleic acid molecule of claim 52.
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.
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.
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.
A pharmaceutical combination comprising the isolated antigen binding protein of any one of claims 1-49 and an immune checkpoint inhibitor.
The drug combination according to claim 57, wherein the immune checkpoint inhibitor comprises a substance that inhibits PD-1/PD-L1 interaction.
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.
The pharmaceutical combination according to any one of claims 57-59, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The pharmaceutical combination according to any one of claims 60-70, wherein the anti-PD-1 antibody comprises pembrolizumab.
The pharmaceutical combination according to any one of claims 57-71, wherein the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The pharmaceutical combination according to any one of claims 72-82, wherein the anti-PD-L1 antibody comprises atezolizumab.
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.
A detection kit for Siglec15, comprising the isolated antigen-binding protein of any one of claims 1-49 or the polypeptide of claim 50.
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.
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.
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.
The use according to claim 88, wherein the disease or condition comprises a tumor.
The use according to any one of claims 88-89, wherein the tumor comprises a tumor associated with the expression of Siglec15.
The use according to any one of claims 89-90, wherein the tumor comprises a solid tumor.
The use according to any one of claims 89-91, wherein the tumor comprises colon cancer.