WO2022223004A1

WO2022223004A1 - Anti-siglec-15 antibody and use thereof

Info

Publication number: WO2022223004A1
Application number: PCT/CN2022/088241
Authority: WO
Inventors: 廖雪梅; 栾珊珊; 杨松霖; 陈娜; 施伟军; 王瓅; 刘颖颖; 李贵祥; 刘婷婷; 王小迪; 夏广新; 柯樱
Original assignee: 上海医药集团股份有限公司
Priority date: 2021-04-22
Filing date: 2022-04-21
Publication date: 2022-10-27
Also published as: CN117203241A

Abstract

The present application relates to an anti-Siglec-15 antibody and the use thereof. The anti-Siglec-15 antibody specifically binds to the human Siglec-15 protein with a K_D value of approximately 4E-09M or less. The present application also provides an immunoconjugate comprising the anti-Siglec-15 antibody, a method for preparing the anti-Siglec-15 antibody, and the use of the anti-Siglec-15 antibody.

Description

Anti-Siglec15 antibody and its use

technical field

The present application relates to the field of biomedicine, in particular to an anti-Siglec15 antibody and use thereof.

Background technique

Siglec15 is a type I transmembrane protein comprising 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, and its function is associated with osteoclast differentiation and bone remodeling. Siglec15 mRNA expression levels are extremely low in most normal human tissues and various immune cell subsets, but high in macrophages.

In 2019, Chen et al. found that siglec15 is a macrophage-related T cell inhibitory molecule whose expression is usually limited to myeloid cells, but is 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, indicating the safety of Siglec15 targets. Siglec15 inhibits antigen-specific T cell responses by inhibiting IL-10. At the same time, the secretion of CD8+ T cells, NK cells and cytokines in Siglec15 knockout mice is increased, the incidence of tumors is decreased, and the survival rate is increased. The above results suggest that Siglec15 may be a very potential tumor therapy target.

At present, the known anti-Siglec-15 antibodies still have defects such as weak affinity and weak anti-tumor effect in vivo. Therefore, it is necessary to develop novel anti-Siglec15 antigen-binding proteins with high affinity and specificity for Siglec15 protein and better anti-tumor effect.

SUMMARY OF THE INVENTION

The application provides an isolated antigen-binding protein, which has one or more of the following properties: 1) in Biacore detection, specifically binds to human Siglec15 protein with a KD value of about 4E-09M or less; 2) In flow assay, it specifically binds to human Siglec15 expressed on the surface of CHOK1 cells with an EC50 value of about 0.1 μg/ml or less; 3) Relieves the proliferation inhibition of PBMC in blood by Siglec15; 4) In ELISA assay , specifically binds to human Siglec15 with an EC50 value of about 0.2 μg/ml or less; 5) specifically binds to monkey Siglec15 with an EC50 value of about 0.1 μg/ml or less in an ELISA assay; and 6) in an ELISA assay , specifically binds to murine Siglec15 with an EC50 value of about 0.1 μg/ml or less.

In certain embodiments, the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:13.

In certain embodiments, the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9.

In certain embodiments, the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; the The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises SEQ ID NO : amino acid sequence shown in 64.

In certain embodiments, the H-FR1 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.

In certain 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: 65 amino acid sequence.

In certain embodiments, the H-FR2 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5-8.

In certain 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: 66 amino acid sequence.

In certain embodiments, the H-FR3 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.

In certain embodiments, the isolated antigen binding protein 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 SEQ ID NO : amino acid sequence shown in 67.

In certain embodiments, the H-FR4 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 14 and 15.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 64; the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:65; the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:66; and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:67.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprising any one of SEQ ID NOs: 1-3 The amino acid sequence of ; the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 5-8; the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 10-12; And the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 14 and 15.

In certain embodiments, the H-FR1, H-FR2, H-FR3 and H-FR4 in the isolated antigen binding protein comprise an amino acid sequence selected from any of the following groups:

a) H-FR1: SEQ ID NO: 1, H-FR2: SEQ ID NO: 5, H-FR3: SEQ ID NO: 10 and H-FR4: SEQ ID NO: 14;

b) H-FR1: SEQ ID NO: 2, H-FR2: SEQ ID NO: 6, H-FR3: SEQ ID NO: 11 and H-FR4: SEQ ID NO: 15;

c) H-FR1: SEQ ID NO: 2, H-FR2: SEQ ID NO: 7, H-FR3: SEQ ID NO: 12 and H-FR4: SEQ ID NO: 15;

d) H-FR1: SEQ ID NO:3, H-FR2: SEQ ID NO:8, H-FR3: SEQ ID NO:12 and H-FR4: SEQ ID NO:15.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO:72.

In certain embodiments, the VH in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 29-32.

In certain embodiments, the isolated antigen binding protein comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:26.

In certain embodiments, the isolated antigen binding protein comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:22.

In certain embodiments, the isolated antigen binding protein comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:19.

In certain embodiments, the isolated antigen binding protein comprises a light chain variable region VL, the VL comprises the LCDR1, LCDR2 and LCDR3, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26; the The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 22; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19.

In certain embodiments, the isolated antigen binding protein comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises SEQ ID NO : amino acid sequence shown in 68.

In certain embodiments, the L-FR1 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 16-18.

In certain embodiments, the isolated antigen-binding protein comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO: 69 amino acid sequence.

In certain embodiments, the L-FR2 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20 and 21.

In certain embodiments, the isolated antigen binding protein comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO: 70 amino acid sequence.

In certain embodiments, the L-FR3 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23-25.

In certain embodiments, the isolated antigen binding protein comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises SEQ ID NO : amino acid sequence shown in 71.

In certain embodiments, the L-FR4 in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27 and 28.

In certain embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 68; the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:69; the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:70; and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:71.

In certain embodiments, the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 comprising the set forth in any one of SEQ ID NOs: 16-18 The amino acid sequence of ; the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20 and 21; the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 23-25; And the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 27 and 28.

In certain embodiments, the L-FR1, L-FR2, L-FR3 and L-FR4 in the isolated antigen binding protein comprise an amino acid sequence selected from any of the following groups:

a) L-FR1: SEQ ID NO: 16, L-FR2: SEQ ID NO: 20, L-FR3: SEQ ID NO: 23 and L-FR4: SEQ ID NO: 27;

b) L-FR1: SEQ ID NO: 17, L-FR2: SEQ ID NO: 21, L-FR3: SEQ ID NO: 24 and L-FR4: SEQ ID NO: 28;

c) L-FR1: SEQ ID NO: 18, L-FR2: SEQ ID NO: 21, L-FR3: SEQ ID NO: 25 and L-FR4: SEQ ID NO: 28;

d) L-FR1: SEQ ID NO: 18, L-FR2: SEQ ID NO: 20, L-FR3: SEQ ID NO: 25 and L-FR4: SEQ ID NO: 28.

In certain embodiments, the isolated antigen binding protein comprises a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO:73.

In certain embodiments, the VL in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 33-36.

In certain embodiments, the VH and VL in the isolated antigen binding protein comprise amino acid sequences selected from any of the following groups:

a) VH: SEQ ID NO: 29 and VL: SEQ ID NO: 33;

b) VH: SEQ ID NO:30 and VL: SEQ ID NO:34;

c) VH: SEQ ID NO:30 and VL: SEQ ID NO:35;

d) VH: SEQ ID NO:30 and VL: SEQ ID NO:36;

e) VH: SEQ ID NO: 31 and VL: SEQ ID NO: 34;

f) VH: SEQ ID NO:31 and VL: SEQ ID NO:35;

g) VH: SEQ ID NO: 31 and VL: SEQ ID NO: 36;

h) VH: SEQ ID NO: 32 and VL: SEQ ID NO: 34;

i) VH: SEQ ID NO:32 and VL: SEQ ID NO:35;

j) VH: SEQ ID NO:32 and VL: SEQ ID NO:36.

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 or an IgY-derived constant region.

In certain embodiments, the heavy chain constant region in the isolated antibody binding protein comprises a constant region derived from human IgG.

In certain embodiments, the heavy chain constant region in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39-42.

In certain embodiments, the isolated antigen binding protein comprises a light chain constant region, and the light chain constant region comprises an Igκ-derived constant region or an Igλ-derived constant region.

In certain embodiments, the light chain constant region in the isolated antigen binding protein comprises a constant region derived from human Igκ.

In certain embodiments, the light chain constant region in the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:43.

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

In certain embodiments, the antigen-binding fragment in 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, the antibody in the isolated antigen binding protein is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In another aspect, the application provides a polypeptide comprising the isolated antigen binding protein.

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

In another aspect, the application provides an isolated nucleic acid molecule encoding the isolated antigen binding protein, or the polypeptide.

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

In another aspect, the application provides a cell comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule and/or the carrier.

In another aspect, the present application provides a method for preparing the isolated antigen-binding protein or the polypeptide, the method comprising culturing the isolated antigen-binding protein or the polypeptide under conditions that allow the isolated antigen-binding protein or the polypeptide to be expressed cell.

In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and/or pharmaceutically acceptable adjuvants and/or excipients.

In another aspect, the application provides a pharmaceutical combination comprising the isolated antigen binding protein and an immune checkpoint inhibitor.

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

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

In certain embodiments, the anti-PD-1 antibody comprises a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:46.

In certain embodiments, the anti-PD-1 antibody comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:45.

In certain embodiments, the anti-PD-1 antibody comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:44.

In certain embodiments, the anti-PD-1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 46; the HCDR2 comprise the amino acid sequence shown in SEQ ID NO:45; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:44.

In certain embodiments, the anti-PD-1 antibody comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO:50.

In certain embodiments, the anti-PD-1 antibody comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:49.

In certain embodiments, the anti-PD-1 antibody comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:48.

In certain embodiments, the anti-PD-1 antibody comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:47.

In certain embodiments, the anti-PD-1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 49; the LCDR2 comprise the amino acid sequence shown in SEQ ID NO:48; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:47.

In certain embodiments, the anti-PD-1 antibody comprises a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO:51.

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 set forth in SEQ ID NO:60.

In certain embodiments, the anti-PD-L1 antibody comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:59.

In certain embodiments, the anti-PD-L1 antibody comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:58.

In certain embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 60; the HCDR2 comprise the amino acid sequence shown in SEQ ID NO:59; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:58.

In certain embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO:54.

In certain embodiments, the anti-PD-L1 antibody comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:63.

In certain embodiments, the anti-PD-L1 antibody comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:62.

In certain embodiments, the anti-PD-L1 antibody comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:61.

In certain embodiments, the anti-PD-L1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 63; the LCDR2 comprise the amino acid sequence shown in SEQ ID NO:62; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:61.

In certain embodiments, the anti-PD-L1 antibody comprises a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO:55.

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

In certain embodiments, the pharmaceutical combination may be a pharmaceutical composition.

In another aspect, the present application provides a method for detecting or assaying Siglec15, the method comprising using the isolated antigen binding protein or the polypeptide.

In another aspect, the present application provides a Siglec15 detection kit, which comprises the isolated antigen-binding protein or the polypeptide.

In another aspect, the present application provides a use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit.

In another aspect, the application provides a method of modulating an immune response, comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the The isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition, and/or the pharmaceutically acceptable therapeutic agent.

In another aspect, the application provides a method of modulating an immune response comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination, and/or a pharmaceutically acceptable therapeutic agent.

In another aspect, the application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, It is used to prevent, alleviate and/or treat a disease or disorder.

In another aspect, the application provides the pharmaceutical combination for the prevention, alleviation and/or treatment of a disease or disorder.

In certain embodiments, the disease or disorder comprises abnormal bone metabolism.

In certain embodiments, the abnormal bone metabolism comprises osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or bone metastases from cancer, bone destruction Loss of tooth mass due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta.

In certain embodiments, the abnormal bone metabolism comprises osteoporosis.

In certain embodiments, the disease or disorder comprises a tumor.

In certain embodiments, the tumor comprises a tumor associated with the expression of Siglec15.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor comprises colon cancer.

In another aspect, the present application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the drug combination Use of the substance in the preparation of a medicament for the prevention and/or treatment of a disease or disorder.

In another aspect, the present application provides the use of the pharmaceutical combination in the manufacture of a medicament for preventing and/or treating a disease or disorder.

In certain embodiments, the abnormal bone metabolism comprises osteoporosis.

In certain embodiments, the disease or disorder comprises a tumor.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor comprises colon cancer.

In another aspect, the present application provides a method of preventing and/or treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate compound, the isolated nucleic acid molecule, the vector, and/or the cell.

In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination.

In certain embodiments, the abnormal bone metabolism comprises osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or bone metastases from cancer, bone destruction Decreased quality of life, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta.

In certain embodiments, the abnormal bone metabolism comprises osteoporosis.

In certain embodiments, the disease or disorder comprises a tumor.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor comprises colon cancer.

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

Description of drawings

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows the husiglec-15-hFc antigenic activity measured by the ELISA described herein.

Figure 2A shows the cyno-siglec-15-his antigenic activity as measured by the ELISA described herein.

Figure 2B shows the husiglec-15-his antigenic activity measured by the ELISA described herein.

Figure 3 shows serum titers from multiple immunized mice.

Figure 4 shows the binding curve of an exemplary Siglec15 antigen binding protein to human Siglec15 expressed on the surface of CHOK1 cells.

Figure 5A shows the effect of the exemplary Siglec15 antigen binding protein described herein on CD8+ T cell proliferation.

Figure 5B shows the effect of the exemplary Siglec15 antigen binding protein described herein on CD4+ T cell proliferation.

Figure 5C shows the effect of the exemplary Siglec15 antigen binding protein described herein on IFN-γ expression.

Figure 6A shows the binding curve of an exemplary humanized Siglec15 binding protein described herein to human Siglec15 expressed on the surface of CHOK1 cells.

Figure 6B shows the binding curve of the exemplary humanized Siglec15 binding protein described herein to human Siglec15 expressed on the surface of CHOK1 cells.

Figure 7A shows the effect of the exemplary humanized Siglec15 antigen binding protein described herein on CD8+ T cell proliferation.

Figure 7B shows the effect of the exemplary humanized Siglec15 antigen binding protein described herein on CD4+ T cell proliferation.

Figure 7C shows the effect of the exemplary humanized Siglec15 antigen binding protein described herein on IFN-γ expression.

Figure 8A shows the binding curve of an exemplary humanized Siglec15 antigen binding protein described herein to human Siglec15 antigen.

Figure 8B shows the binding curve of the exemplary humanized Siglec15 antigen binding protein described herein to the monkey Siglec15 antigen.

Figure 8C shows the binding curve of an exemplary humanized Siglec15 antigen binding protein described herein to murine Siglec15 antigen.

Figure 9A shows the results of the anti-tumor efficacy test of the exemplary anti-Siglec15 antibody Ab0 described in the present application in wild-type C57BL/6N mice.

Figure 9B shows body weight changes in wild-type C57BL/6N mice following administration of an exemplary anti-Siglec15 antibody Ab0.

Figure 10A shows the results of the anti-tumor efficacy test of the exemplary anti-Siglec15 antibodies Abl, Ab6 and Ab7 described in this application in wild-type C57BL/6N mice.

Figure 10B shows body weight changes in wild-type C57BL/6N mice following administration of exemplary anti-Siglec15 antibodies Ab1, Ab6 and Ab7.

Figure 11A shows the results of the anti-tumor efficacy test of the exemplary anti-Siglec15 antibodies Ab0, Ab1, Ab6 and Ab7 described in the present application in human Siglec15 transgenic mice.

Figure 11B shows body weight changes in human Siglec15 transgenic mice following administration of exemplary anti-Siglec15 antibodies AbO, Ab1, Ab6 and Ab7.

Figure 12 shows that humanized Siglec15 antigen binding protein inhibits osteoclast formation.

Figure 13A shows the anti-tumor efficacy of the exemplary Siglec15 antigen-binding proteins Ab1, Ab7 and PD-1 antibodies in combination in human PD-1/PD-L1 transgenic mice.

Figure 13B shows the body weight change of human PD-1/PD-L1 transgenic mice after the exemplary Siglec15 antigen-binding protein Ab1, Ab7 and PD-1 antibody are used in combination.

Figure 14A shows the anti-tumor efficacy of the exemplary Siglec15 antigen-binding proteins Ab1, Ab7 and PD-L1 antibodies in combination in human PD-1/PD-L1 transgenic mice.

Figure 14B shows the body weight change of human PD-1/PD-L1 transgenic mice after the combination of the exemplary Siglec15 antigen binding proteins Ab1, Ab7 and PD-L1 antibody described in the present application.

Detailed ways

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "isolated" generally refers to artificial means obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain 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 isolated of. The term "isolated" does not exclude the admixture of artificial or synthetic materials, nor does it exclude the presence of other impurities that do not affect the activity of the material.

In this application, the term "antigen binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, in this application, the term "antigen-binding protein" may include "antibody" or "antigen-binding fragment". For example, the antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may comprise any molecule comprising an antigen-binding portion thereof. The term "antibody" may include monoclonal antibodies, antibody fragments or antibody derivatives including, but not limited to, murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (eg, scFv ), as well as antigen-binding antibody fragments (eg, Fab, Fab', VHH and (Fab)2 fragments). The term "antibody" may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein. Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain can be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs). Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen (eg, human Siglec15). The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The exact boundaries of the CDRs have been defined differently from system to system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a The residue numbering system is specified, and precise residue boundaries are provided that define the CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that despite large diversity at the amino acid sequence level, certain subsections within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subsections were designated L1 , L2 and L3 or H1, H2 and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs. Other boundaries defining CDRs where the Kabat CDRs overlap have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5): 732-45 (1996)). In addition, other CDRs Boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although they can be shortened or lengthened according to predictions or experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding In this application, the CDRs can be defined by using the Kabat numbering system.

In this application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind an antigen. The antigen-binding function of an antibody can be achieved by full-length fragments of the antibody. Antigen binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2, or alternatively, comprising 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 one-arm; (5) dAb fragment composed of VH domain (Ward et al, (1989) Nature 341:544-546); (6) a combination of separate complementarity determining regions (CDRs) and (7) two or more separate CDRs optionally linked by a linker. For example, a monovalent single-chain molecule Fv (scFv) formed by the pairing of VL and VH may also be included (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883). For example, a class of antibody VHHs that lack the antibody light chain and only have the heavy chain variable region can also be included (for example, see Kang Xiaozhen et al., Chinese Journal of Biological Engineering, 2018, 34(12): 1974-1984). The "antigen-binding portion" may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) with 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 this application, the term "monoclonal antibody" generally refers to a population of substantially homologous antibodies, ie the individual antibodies comprising the population are identical except for possible naturally occurring mutations present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. For example, the monoclonal antibodies can be prepared 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 by 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 portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while The remainder of the chain is then homologous to the corresponding sequence in another species. For example, the variable regions of both light and heavy chains are derived from variable regions of antibodies from one animal species (eg, mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (eg, human) . For example, to obtain a chimeric antibody, non-human B cells or hybridoma cells can be used to generate variable regions, and the constant regions combined therewith are derived from humans. The variable regions have the advantage of being easy to prepare and their specificity is not affected by the source of the constant regions with which they are combined. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of eliciting an immune response when the chimeric antibody is injected is lower than that of using 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 non-human immunoglobulins, thereby reducing the immunogenicity of the xenogeneic antibody when introduced into humans, while at the same time The full antigen-binding affinity and specificity of the antibody is maintained. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof can be used; including "reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), "hyperchimerization", (Queen, et al., 1989 Proc Natl Acad Sci USA 86 : 10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88: 2869-2873; Co, et al., 1992 J Immunol 148: 1149-1154) and "veneering", (Mark, et al ., "Derivation of therapeutically active humanized and veneered anti-CD18antibodies." In: Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), Surface Reconstruction (US Patent US5639641) and other technical means to humanize the non-human binding domain. If other regions, such as hinge and constant region domains, are also derived from non-human sources, these regions can also be humanized.

In this application, the term "murine antibody" generally refers to antibodies in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the present application may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, eg, may include mutations introduced by random or point mutation in vitro or by somatic mutation in vivo.

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 are in use Siglec15 gene transfected cells expressed. In the present application, Siglec15 may be human Siglec15, whose accession number in UniProt/Swiss-Prot is Q6ZMC9. For example, Siglec15 can be a functionally active fragment of human Siglec15. For example, the "functionally active fragment" can include a fragment that retains the endogenous function of at least one naturally occurring protein (eg, binds to the antigen binding proteins described herein). For example, the "functionally active fragment" may include a domain that binds to the antigen-binding protein of the present application.

In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functionally active fragments, derivatives, analogs, homologues, and fragments thereof.

The term "functionally active fragment" refers to a polypeptide having substantially the same amino acid sequence or encoded by substantially the same 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 refers to one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least at least A sequence of endogenous functions. 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 a naturally occurring protein and/or polynucleotide , 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 /or addition, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

In this application, the term "analog" generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie possessing at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics chemical compounds.

Generally, amino acid substitutions, such as at least 1 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutions, can be made, so long as the modified sequence remains substantially as desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

In this application, the term "homologue" generally refers to an amino acid sequence or nucleotide sequence that has some homology to a naturally occurring sequence. The term "homology" may be equivalent to sequence "identity". Homologous sequences can include amino acid sequences that can 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 contain the same active site, etc., as the subject amino acid sequence. Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity. In the present application, a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of. To determine sequence identity, sequence alignments can be performed by various means known to those skilled in the art, eg, 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 among the full-length sequences being compared.

The proteins or polypeptides used in the present 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 based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic properties 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 containing uncharged polar headgroups with similar hydrophilicity values include amino acids Paraparagine, Glutamine, Serine, Threonine and Tyrosine.

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 the 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 protein expression of Siglec15 may be a Siglec15 positive tumor. In Siglec15-positive tumors, the protein expression of Siglec15 on the tumor cell surface or in the tumor microenvironment was approximately 1%, 5%, 10%, 15%, 20%, 25%, 30% higher than normal cells, 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, CT scan, ultrasound, or palpation). For example, the solid tumor can include colon cancer.

In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (eg, covalently via a linker molecule) of the other therapeutic agent to the isolated antigen-binding protein, the conjugation The other therapeutic agent can be delivered to target cells (eg, tumor cells) 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 "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

In this application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.

In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The vector can transfer the inserted nucleic acid molecule into and/or between cells. The vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell. Typically, the vector can produce the desired expression product by culturing suitable cells containing the vector. In the present application, the vector may comprise a lentiviral vector.

In this application, the term "cell" generally refers to a plasmid or vector that can or has contained a nucleic acid molecule described herein, or an individual cell capable of expressing a polypeptide described herein or an antigen binding protein described herein , cell lines or cell cultures. The cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the polypeptides or antigen-binding proteins described herein. The cells can be obtained by transfecting cells in vitro using the vectors described herein. The cells may be prokaryotic cells (eg E. coli) or eukaryotic cells (eg yeast cells, eg 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 can 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 "treating" generally refers to: (i) preventing the occurrence of a disease, disorder or condition in a patient who may be susceptible to a disease, disorder and/or condition but has not been diagnosed with the disease; (ii) inhibiting the disease , disease or condition, i.e. arresting its development; and (iii) alleviating the disease, disorder or condition, i.e. causing the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subsided.

In this application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (eg, binding to labeling components). The term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.

In this application, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to nucleosides of any length Polymeric forms of acids, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation to labeled components.

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 in titration measurements at equilibrium, or Value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka). Association rate constants (ka), dissociation rate constants (kd), and equilibrium dissociation constants (KD) are used to represent binding of a binding protein (eg, the isolated antigen-binding proteins described herein) to antigen (eg, _Siglec15 protein) 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 equilibration in physiological buffers. For example, the KD value can be determined by Biacore (Biomolecular Interaction Analysis) (eg, an instrument available from BIAcore International AB, _aGE Healthcare company, Uppsala, Sweden), or can be detected using other experimental approaches and instruments such as Octet. 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 K _D value can also be determined by an amine coupling kit.

In this application, the term "and/or" should be understood to mean either or both of the alternatives.

In this application, the term "comprising" generally means including the expressly specified features, but not excluding other elements. In certain instances, "comprising" also encompasses including only the specified components. For example, to include is also expressed as also to mean "consisting of."

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%, 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 "comprising" generally refers to the meaning of including, encompassing, containing or encompassing. In some cases, it also means "for" and "consisting of".

Detailed description of the invention

The isolated antigen binding proteins described herein

In one aspect, the application provides an isolated antigen-binding protein that can have a KD value of about 4E- _09M or less in a Biacore assay (eg, the KD is not higher than about 4E- _09M , not higher than About 3.5E-09M, not higher than about 3E-09M, not higher than about 2.5E-09M, not higher than about 2E-09M, not higher than about 1.5E-09M, not higher than about 1E-09M, not higher at about 9E-10M, not higher than about 5E-10M, not higher than about 1E-10M, not higher than about 5E-11M, not higher than about 1E-11M or not higher than 5E-12M or less) with human Siglec15 protein specific binding.

In the present application, the isolated antigen-binding protein can have an EC ₅₀ value of about 0.1 μg/ml or less (eg, the EC50 value is not higher than about 0.1 μg/ml, not higher than About 99ng/ml, no more than about 95ng/ml, no more than about 90ng/ml, no more than about 85ng/ml, no more than about 80ng/ml, no more than about 75ng/ml, no more than about 70ng /ml, not higher than about 65 ng/ml, not higher than about 60 ng/ml, not higher than about 55 ng/ml, or not higher than about 50 ng/ml or less) specifically binds to human Siglec15 expressed on CHOK1 cells.

In the present application, the isolated antigen-binding protein can have an EC ₅₀ value of about 0.2 μg/ml or less in an ELISA assay (eg, the EC ₅₀ value is not higher than about 0.2 μg/ml, not higher than About 0.1 μg/ml, no more than about 99 ng/ml, no more than about 95 ng/ml, no more than about 90 ng/ml, no more than about 85 ng/ml, no more than about 80 ng/ml, no more than about 75ng/ml, not higher than about 70ng/ml, not higher than about 65ng/ml, not higher than about 60ng/ml, not higher than about 55ng/ml or not higher than about 50ng/ml or less) specific for human Siglec15 sexual union.

In the present application, the isolated antigen-binding protein can have an EC ₅₀ value of about 0.1 μg/ml or less in an ELISA assay (eg, the EC ₅₀ value is not higher than about 0.1 μg/ml, not higher than About 99ng/ml, no more than about 95ng/ml, no more than about 90ng/ml, no more than about 85ng/ml, no more than about 80ng/ml, no more than about 75ng/ml, no more than about 70ng /ml, no more than about 65 ng/ml, no more than about 60 ng/ml, no more than about 55 ng/ml, or no more than about 50 ng/ml or less) specifically binds to monkey Siglec15.

In the present application, the isolated antigen-binding protein can have an EC ₅₀ value of about 0.1 μg/ml or less in an ELISA assay (eg, the EC ₅₀ value is not higher than about 0.1 μg/ml, not higher than About 99ng/ml, no more than about 95ng/ml, no more than about 90ng/ml, no more than about 85ng/ml, no more than about 80ng/ml, no more than about 75ng/ml, no more than about 70ng /ml, no more than about 65 ng/ml, no more than about 60 ng/ml, no more than about 55 ng/ml, or no more than about 50 ng/ml or less) specifically binds to murine Siglec15.

In the present application, the isolated antigen binding protein can relieve the proliferation inhibition of PBMC in blood by Siglec15. In the present application, the isolated antigen binding protein can promote the proliferation of CD8 and CD4 T cells. In the present application, the isolated antigen binding protein can promote the expression of IFN-γ.

In one aspect, the application provides an isolated antigen binding protein, which can comprise at least one CDR in the VH of the variable region of the antibody heavy chain, and the VH can comprise the amino acid sequence shown in SEQ ID NO:72.

For example, the VH may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 29-32. In the present application, the HCDR of the isolated antigen-binding protein can be divided in any form, as long as the VH is the same as the amino acid sequence shown in any one of SEQ ID NOs: 29-32, the HCDR obtained by dividing in any form can be fall within the scope of protection of this application.

The CDRs of antibodies, also known as complementarity determining regions, are part of the variable region. Amino acid residues in this region can make contact with the antigen or antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, Kabat/Chothia, etc. in combination. These coding systems are known in the art, see eg http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR regions. In the present application, the CDRs encompass CDR sequences that are divided according to any CDR division; variants thereof are also encompassed, the variants comprising substitution, deletion and/or addition of one or more amino acids to the amino acid sequence of the CDR . For example 1-30, 1-20 or 1-10, also for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or Or insertions; homologues thereof are also encompassed, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 92%, amino acid sequences of 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 Kabat 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 HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the HCDR3 sequence of the antigen binding protein can be defined according to the Kabat coding system.

In the present application, the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the HCDR2 sequence of the antigen binding protein can be defined according to the Kabat coding system.

In the present application, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:4. For example, the HCDRl sequence of the antigen binding protein can be defined according to the Kabat coding system.

For example, the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13 amino acid sequence shown. For example, the antigen binding protein can include antibodies AbO to Ab9 or antigen binding fragments having the same HCDR3 therewith (eg, having the same HCDR1-3).

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

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:64. For example, H-FR1 of the antigen binding protein has an amino acid substitution (eg, conservative amino acid substitution, etc.) at one or more amino acids selected from the group compared to the sequence shown in SEQ ID NO: 64: X ₂ , X ₉ , X ₁₆ , X ₁₇ and X ₂₀ .

Q X ₂ QLVQSG X ₉ ELKKPG X ₁₆ X ₁₇ VK X ₂₀ SCKASGYTFT (SEQ ID NO: 64), wherein X ₂ can be I or V, X ₉ can be P or S, X ₁₆ can be A or E, X ₁₇ Can be T or S, X ₂₀ can be I or V.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

In the present application, the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:65. For example, H-FR2 of the antigen-binding protein has an amino acid substitution (eg, conservative amino acid substitution, etc.) at one or more amino acids selected from the group compared to the sequence shown in SEQ ID NO: 65: X ₃ , X ₈ and X ₁₁ .

WV X ₃ QAPG X ₈ GL X ₁₁ WMG (SEQ ID NO: 65), wherein X ₃ can be K or R, X ₈ can be K or Q, and X ₁₁ can be E or K.

In the present application, the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-8.

In the present application, the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:66. For example, H-FR3 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 compared to the sequence shown in SEQ ID NO: 66: X ₃ , X ₇ , X ₁₀ , X ₁₂ , X ₁₈ , X ₁₉ , X ₂₂ , X ₂₇ and X ₂₉ .

RF X ₃ FSL X ₇ TS X ₁₀ S X ₁₂ AYLQI X ₁₈ X ₁₉ LK X ₂₂ EDTA X ₂₇ Y X ₂₉ CAR (SEQ ID NO: 66), wherein X ₃ can be A or V and X ₇ can be D or E , X ₁₀ can be A or V, X ₁₂ can be M or T, X ₁₈ can be N or S, X ₁₉ can be N or S, X ₂₂ can be A or N, X ₂₇ can be T or V and X ₂₉ can be F or Y.

In the present application, the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10-12.

In the present application, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:67. For example, H-FR4 of the antigen binding protein has an amino acid substitution (eg, conservative amino acid substitution, etc.) at one or more amino acids selected from the group compared to the sequence shown in SEQ ID NO: 67: X ₆ and X ₇ .

WGQGT X ₆ X ₇ TVSS (SEQ ID NO: 67), wherein X ₆ can be L or T and X ₇ can be L or V.

In the present application, the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 14 and 15.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 64; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 65; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:66; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:67.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-3; the H-FR2 may comprise any one of SEQ ID NOs: 5-8 The amino acid sequence shown in item; the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10-12; and the H-FR4 may comprise any one of SEQ ID NOs: 14 and 15 amino acid sequence shown.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 10; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14. For example, the antigen-binding protein may comprise antibody Ab0 or an antigen-binding fragment thereof having the same H-FR1-4.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 6; the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 11; and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the antigen-binding protein may include antibodies Ab1, Ab2, Ab3, or antigen-binding fragments thereof having the same H-FR1-4.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 7; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 12; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the antigen binding protein may comprise antibodies Ab4, Ab5, Ab6 or antigen binding fragments thereof having the same H-FR1-4.

In the present application, the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 3; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 12; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the antigen binding protein may comprise antibodies Ab7, Ab8, Ab9 or antigen binding fragments thereof having the same H-FR1-4.

In the present application, the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:72. For example, the antigen-binding protein comprises a VH having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: _X2 , _X9 , _X16 , _X17 , _X20 , _X38 , _X43 , _X46 , _X69 , _X73 , _X76 , _X78 , _X84 , _X85 , _X88 , _X93 , _X95 , X ₁₁₅ and X ₁₁₆ .

Q X ₂ QLVQSG X ₉ ELKKPG X ₁₆ X ₁₇ VK X ₂₀ SCKASGYTFTTYEMSWV X ₃₈ QAPG X ₄₃ GL X ₄₆ WMGWINTYSGVPTYADDFKGRF X ₆₉ FSL X ₇₃ TS X ₇₆ S X ₇₈ AYLQI X ₈₄ X ₈₅ LK X ₈₈ EDTA X ₉₃ Y X ₉₅ _{CAREGVYGTDYPYFDS} ₁₁₆ TVSS (SEQ ID NO: 72), wherein X ₂ can be I or V, X ₉ can be P or S, X ₁₆ can be A or E, X ₁₇ can be T or S, X ₂₀ can be I or V, X ₃₈ can be K or R, X ₄₃ can be K or Q, X ₄₆ can be E or K, X ₆₉ can be A or V, X ₇₃ can be D or E, X ₇₆ can be A or V, X ₇₈ can be M or T, X ₈₄ can be N or S, X ₈₅ can be N or S, X ₈₈ can be A or N, X ₉₃ can be T or V and X ₉₅ can be F or Y, X ₁₁₅ Can be L or T, X ₁₁₆ can be L or V.

In the present application, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 29-32.

In the present application, the antigen binding protein may comprise a heavy chain constant region, which may include an IgG-derived constant region or an IgY-derived constant region.

In the present application, the heavy chain constant region may include a constant region derived from IgGl, IgG2, IgG3 or IgG4.

For example, the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 39-42.

In the present application, the antigen binding protein may comprise at least one CDR in the variable region VL of the antibody light chain, and the VL may comprise the amino acid sequence shown in SEQ ID NO:73.

For example, the VL may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 33-36. In the present application, the LCDR of the isolated antigen-binding protein can be divided in any form, as long as the VL is the same as the amino acid sequence shown in any one of SEQ ID NOs: 33-36, the LCDR obtained by dividing in any form can be fall within the scope of protection of this application.

In the present application, the antigen binding protein may comprise a light chain variable region VL, and the VL may comprise at least one, at least two or at least three of LCDR1, LCDR2 and LCDR3.

In the present application, the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the LCDR3 of the antigen binding protein can be defined according to the Kabat numbering system.

In the present application, the LCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 22. For example, the LCDR2 of the antigen binding protein can be defined according to the Kabat numbering system.

In the present application, the LCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 19. For example, the LCDR1 of the antigen binding protein can be defined according to the Kabat numbering system.

For example, LCDR1 of the antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; and the LCDR3 may comprise SEQ ID NO: The amino acid sequence shown in 26. For example, the antigen binding protein can include antibodies Ab0 to Ab9 or antigen binding fragments that have the same LCDR3 (eg, have the same LCDR1-3).

For example, the VL of the antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:68. For example, L-FR1 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 compared to the sequence shown in SEQ ID NO: 68: X ₅ , X ₁₅ , X ₁₈ and X ₁₉ .

DIQM X ₅ QSPSSLSAS X ₁₅ GD X ₁₈ X ₁₉ TITC (SEQ ID NO: 68), wherein X ₅ can be N or T, X ₁₅ can be L or V, X ₁₈ can be R or T and X ₁₉ can be I or V.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 16-18.

In the present application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:69. For example, the L-FR2 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 compared to the sequence shown in SEQ ID NO: 69: X ₈ and X ₉ .

WYQQKPG X ₈ X ₉ PKLLIY (SEQ ID NO: 69), wherein X ₈ can be K or N and X ₉ can be A or I.

In the present application, the L-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 20 and 21.

In the present application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:70. For example, L-FR3 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 compared to the sequence shown in SEQ ID NO: 70: X ₁₂ , X ₁₄ and X ₂₇ .

GVPSRFSGSGS X ₁₂ T X ₁₄ FTLTISSLQPED X ₂₇ ATYYC (SEQ ID NO: 70), wherein X ₁₂ can be G or R, X ₁₄ can be D or G and X ₂₇ can be F or I.

In the present application, the L-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 23-25.

In the present application, the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:71. For example, L-FR4 of the antigen binding protein has an amino acid substitution (eg, conservative amino acid substitution, etc.) at one or more amino acids selected from the group compared to the sequence shown in SEQ ID NO: 71: X ₇ .

_FGGGTK X7EIK (SEQ ID NO: ₇₁ ), wherein X7 can be L or V.

In the present application, the L-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 27 and 28.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 68; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 16-18; the L-FR2 may comprise any one of SEQ ID NOs: 20 and 21 The amino acid sequence shown in item; the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 23-25; and the L-FR4 may comprise any one of SEQ ID NOs: 27-28 amino acid sequence shown.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 16; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 20; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 23; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 27. For example, the antigen binding protein may comprise antibody Ab0 or an antibody having the same L-FR1-4 therewith.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 17; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 24; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the antigen binding protein may include antibodies Ab1, Ab4, Ab7 or antibodies having the same H-FR1-4 therewith.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the antigen binding protein may include antibodies Ab2, Ab5, Ab8 or antibodies having the same L-FR1-4 therewith.

In the present application, the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 20; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the antigen binding protein may comprise antibodies Ab3, Ab6, Ab9 or antibodies having the same H-FR1-4 therewith.

In the present application, the antigen binding protein may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:73. For example, the VL of the antigen binding protein has an amino acid substitution (eg, conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X ₅ , X compared to the sequence shown in SEQ ID NO: 73 ₁₅ , X ₁₈ , X ₁₉ , X ₄₂ , X ₄₃ , X ₆₈ , X ₇₀ , X ₈₃ , X ₁₀₄ .

DIQMX ₅ QSPSSLSASX ₁₅ GDX ₁₈ X ₁₉ TITCHASQNINVWLSWYQQKPGX ₄₂ X ₄₃ PKLLIYKASNLHTGVPSRFSGSGSX ₆₈ TX ₇₀ FTLTISSLQPEDX ₈₃ ATYYCQQGQSSPLTFGGGTKX ₁₀₄ EIK

(SEQ ID NO: 73), wherein X ₅ can be N or T, X ₁₅ can be L or V, X ₁₈ can be R or T, X ₁₉ can be I or V, X ₄₂ can be K or N, X ₄₃ can be A or I, X ₆₈ can be G or R, X ₇₀ can be D or G, X ₈₃ can be F or I, and X ₁₀₄ can be L or V.

In the present application, the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 33-36.

In the present application, the antigen binding protein may comprise a light chain constant region, which may include an Igκ-derived constant region or an Igλ-derived constant region.

For example, the light chain constant region can include a constant region derived from IgK.

For example, the light chain constant region of the antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID NO:43.

In the present application, the antigen binding protein may comprise HCDR1-3 and LCDR1-3. For example, the HCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:4; the HCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:9; the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:9; comprise the amino acid sequence shown in SEQ ID NO: 13; the LCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 of the antigen-binding protein may comprise the amino acid shown in SEQ ID NO: 22 sequence; LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:26. For example, the antigen-binding protein can include antibodies Ab0-Ab9 or antigen-binding fragments thereof having the same HCDR3 (e.g., having the same HCDR1-3) and LCDR3 (e.g., having the same LCDR1-3).

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:1; the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:10 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 16; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 16 : the amino acid sequence shown in 20; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 23; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 27. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:29. For example, the antigen binding protein may comprise antibody AbO or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:33. For example, the antigen binding protein may comprise antibody AbO or an antigen binding protein having the same light chain variable region.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:6; the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:11 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 17; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 17 : the amino acid sequence shown in 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 24; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the antigen binding protein may include antibody Ab1 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:34. For example, the antigen binding protein may comprise antibody Ab1 or an antigen binding protein having the same light chain variable region therewith.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:6; the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:11 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 18 : the amino acid sequence shown in 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the antigen binding protein may include antibody Ab2 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:35. For example, the antigen binding protein may include antibody Ab2 or an antigen binding protein having the same light chain variable region therewith.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:6; the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:11 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 18 : the amino acid sequence shown in 20; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the antigen binding protein may comprise the antigen binding fragment Ab3 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:36. For example, the antigen binding protein may comprise antibody Ab3 or an antigen binding protein having the same light chain variable region.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO:2; the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO:7; the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO:12 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 17; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 17 : the amino acid sequence shown in 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 24; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:31. For example, the antigen binding protein may include antibody Ab4 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:34. For example, the antigen binding protein may include antibody Ab4 or an antigen binding protein having the same light chain variable region.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4. The light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO:2; the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO:7; the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO:12 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 18 : the amino acid sequence shown in 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:31. For example, the antigen binding protein may comprise antibody Ab5 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:35. For example, the antigen binding protein may comprise antibody Ab5 or an antigen binding protein having the same light chain variable region therewith.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO:2; the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO:7; the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO:12 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 18 : the amino acid sequence shown in 20; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:31. For example, the antigen binding protein may comprise antibody Ab6 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:36. For example, the antigen binding protein may comprise antibody Ab6 or an antigen binding protein having the same light chain variable region therewith.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO:3; the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO:8; the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO:12 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 17; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 17 : the amino acid sequence shown in 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 24; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:32. For example, the antigen binding protein may comprise antibody Ab7 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:34. For example, the antigen binding protein may comprise antibody Ab7 or an antigen binding protein having the same light chain variable region.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO:3; the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO:8; the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO:12 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 18 : the amino acid sequence shown in 21; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:32. For example, the antigen binding protein may comprise antibody Ab8 or an antigen binding protein having the same heavy chain variable region therewith. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:35. For example, the antigen binding protein may comprise antibody Ab8 or an antigen binding protein having the same light chain variable region therewith.

In the present application, the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4. The light chain variable region may comprise LCDR1-3 and L-FR1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO:3; the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO:8; the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO:12 The amino acid sequence shown; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15; the L-FR1 may comprise the amino acid sequence of SEQ ID NO: 18; the L-FR2 may comprise the amino acid sequence of SEQ ID NO: 18 : the amino acid sequence shown in 20; the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 25; the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28. For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:32. For example, the antigen binding protein may comprise antibody Ab9 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:36. For example, the antigen binding protein can include antibody Ab9 or an antigen binding protein having the same light chain variable region.

In the present application, the isolated antigen binding protein may also compete for binding to the human Siglec15 protein with a reference antibody, which may comprise a heavy chain variable region VH, which may comprise HCDR1, HCDR2 and HCDR3 at least one, two, or three of them.

In the present application, the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the sequence of the HCDR3 of the reference antibody can be defined according to the Kabat coding system.

In the present application, the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the sequence of the HCDR2 of the reference antibody can be defined according to the Kabat coding system.

In the present application, the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:4. For example, the sequence of the HCDR1 of the reference antibody can be defined according to the Kabat coding system.

For example, HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13 amino acid sequence shown. For example, the reference antibody can include antibodies Ab0 to Ab9 or antigen binding proteins that have the same HCDR3 (eg, have the same HCDR1-3).

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:72.

In the present application, the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NOs: 29-32.

In the present application, the reference antibody may comprise a heavy chain constant region, which may include an IgG-derived constant region or an IgY-derived constant region.

For example, the heavy chain constant region of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 39-42.

In the present application, the reference antibody may comprise a light chain variable region VL, which may comprise LCDR1, LCDR2 and LCDR3.

In the present application, the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:26. For example, the sequence of LCDR3 of the reference antibody can be defined according to the Kabat coding system.

In the present application, the LCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 22. For example, the sequence of LCDR2 of the reference antibody can be defined according to the Kabat coding system.

In the present application, the LCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 19. For example, the sequence of LCDR1 of the reference antibody can be defined according to the Kabat coding system.

For example, LCDR1 of the reference antibody described herein may comprise the amino acid sequence set forth in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 22; and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 22; The amino acid sequence shown in 26. For example, the reference antibody can include antibodies AbO to Ab9 or antigen binding proteins that have the same LCDR3 (eg, have the same LCDR1-3).

In the present application, the reference antibody may comprise a light chain variable region, and the light chain variable region may comprise the amino acid sequence shown in SEQ ID NO:73.

DIQMX ₅ QSPSSLSASX ₁₅ GDX ₁₈ X ₁₉ TITCHASQNINVWLSWYQQKPGX ₄₂ X ₄₃ PKLLIYKASNLHTGVPSRFSGSGSX ₆₈ TX ₇₀ FTLTISSLQPEDX ₈₃ _{ATYYCQQGQSSPLTFGGGTKX} ₁₀₄ EIK (SEQ ID NO: 73), where X ₅ can be N or T, X ₁₅ can be L or V, is R or T, X ₁₉ can be I or V, X ₄₂ can be K or N, X ₄₃ can be A or I, X ₆₈ can be G or R, X ₇₀ can be D or G, X ₈₃ can be F or I, X ₁₀₄ can be L or V.

In the present application, the light chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NOs: 33-36.

In the present application, the reference antibody may comprise a light chain constant region comprising a constant region derived from Igκ or a constant region derived from Igλ.

For example, the light chain constant region of the reference antibody comprises the amino acid sequence set forth in any one of SEQ ID NO:43.

In the present application, the reference antibody may comprise HCDR1-3 and LCDR1-3. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:13; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 22; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 26. For example, the reference antibody can include antibodies Ab0-Ab9 or antigen binding proteins that have the same HCDR3 (eg, have the same HCDR1-3) and LCDR3 (eg, have the same LCDR1-3).

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:29. For example, the reference antibody may comprise antibody AbO or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:33. For example, the reference antibody may comprise antibody AbO or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab0 or an antigen binding protein having the same heavy chain variable region and light chain variable region.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the reference antibody may comprise antibody Ab1 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:34. For example, the reference antibody may comprise antibody Ab1 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab1 or an antigen binding protein having the same heavy chain variable region and light chain variable region.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the reference antibody may comprise antibody Ab2 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:35. For example, the reference antibody may comprise antibody Ab2 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab2 or an antigen binding protein having the same heavy chain variable region and light chain variable region.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:30. For example, the reference antibody may comprise antibody Ab3 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:36. For example, the reference antibody may comprise antibody Ab3 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab3 or an antigen binding protein having the same heavy chain variable region and light chain variable region.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:31. For example, the reference antibody may comprise antibody Ab4 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:34. For example, the reference antibody may comprise antibody Ab4 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab4 or an antigen binding protein having the same heavy and light chain variable regions.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:31. For example, the reference antibody may comprise antibody Ab5 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:35. For example, the reference antibody may comprise antibody Ab5 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab5 or an antigen binding protein having the same heavy chain variable region and light chain variable region.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:31. For example, the reference antibody may comprise antibody Ab6 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:36. For example, the reference antibody may comprise antibody Ab6 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab6 or an antigen binding protein having the same heavy and light chain variable regions.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:32. For example, the reference antibody may comprise an antigen binding protein with which antibody Ab7 has the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:34. For example, the reference antibody may comprise antibody Ab7 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may include antibody Ab7 or an antigen binding protein having the same heavy and light chain variable regions.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:32. For example, the reference antibody may comprise antibody Ab8 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:35. For example, the reference antibody may comprise antibody Ab8 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may include antibody Ab8 or an antigen binding protein having the same heavy and light chain variable regions.

In the present application, the reference antibody may comprise a heavy chain variable region and a light chain variable region. For example, the heavy chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:32. For example, the reference antibody may comprise antibody Ab9 or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:36. For example, the reference antibody may comprise antibody Ab9 or an antigen binding protein having the same light chain variable region. For example, the reference antibody may comprise antibody Ab9 or an antigen binding protein having the same heavy and light chain variable regions.

Polypeptides and Immunoconjugates

In another aspect, the application provides one or more polypeptides that may comprise the isolated antigen binding proteins of the application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptides can include multispecific antibodies (eg, bispecific antibodies).

In another aspect, the application provides one or more immunoconjugates that can comprise the isolated antigen binding proteins of the application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, marker and/or detection agent.

Nucleic Acids, Vectors and Cells

In another aspect, the present application also provides isolated one or more nucleic acid molecules that encode the isolated antigen-binding proteins described herein. For example, each of the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode a portion thereof (eg, HCDR1-3, one of the heavy chain variable regions, or variety).

For example, when the nucleic acid molecules separately encode a portion of the antigen-binding protein, the products encoded by the nucleic acid molecules together can form a functional (eg, can bind Siglec15) isolated antigen-binding protein of the present application.

The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.

In the present application, nucleic acids encoding the isolated antigen-binding proteins can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the isolated antigen-binding proteins described herein protein nucleic acid molecules.

In another aspect, the application provides one or more vectors comprising one or more nucleic acid molecules described herein. One or more of the nucleic acid molecules may be included in each vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host. Such control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc. For example, a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters may include, for example, the promoters for SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's A portion may be fused to a portion of the gene promoter for other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns. One or more nucleic acid molecules described herein can be operably linked to the expression control element.

The vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering. For example, the vector can be an expression vector. For example, the vector can be a viral vector. The viral vector can be administered directly to the patient (in vivo) or can be administered in an indirect form, eg, by treating cells with the virus in vitro, and then administering the treated cells to the patient (ex vivo). Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology. Conventional virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex vectors for gene transfer. In some cases, retroviral, lentiviral, and adeno-associated virus methods can be used to integrate gene transfer into the host genome, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise long terminal repeats 5'LTR and truncated 3'LTRs, RREs, rev response elements (cPPT), central termination sequences (CTS) and/or post-translational regulatory elements (WPRE). The vectors described herein can be introduced into cells.

In another aspect, the application provides a cell. The cells may comprise the isolated antigen binding proteins described herein, the polypeptides, the immunoconjugates, one or more nucleic acid molecules and/or one or more vectors described herein . For example, each or each cell may contain one or one nucleic acid molecule or vector described herein. For example, each or each cell can comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into such host cells, such as prokaryotic cells (eg, bacterial cells), CHO cells, NS/0 cells, HEK293T cells, 293F cells, or HEK293A cells, or other eukaryotic cells, Such as cells from plants, fungi or yeast cells, etc. The vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like. For example, the cells can include yeast cells. For example, the cells can include E. coli cells. For example, the cells can include mammalian cells. For example, the cells can include immune cells.

The cells may include immune cells. In certain instances, the cells can include immune cells. For example, the cells can include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells cell.

Pharmaceutical Compositions and Drug Combinations

In another aspect, the application provides a pharmaceutical composition. The pharmaceutical composition may comprise the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and/or the isolated antigen-binding protein described herein. or pharmaceutically acceptable adjuvants and/or excipients. In the present application, the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional medium or agent is 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 excipients. For example, the excipients may include binders, fillers, disintegrants, lubricants in the tablet. For example, the excipients may include wine, vinegar, medicinal juice, etc. in Chinese medicine pills. For example, the excipient may comprise a base part of a semisolid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavors, fragrances, solubilizers, emulsifiers, solubilizers, osmo-regulators, colorants in liquid formulations.

In the present 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 blocker may 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 may be selected from the group consisting of Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalizumab) ), Sintilimab (sintilimab) and Tislelizumab (tislelizumab). For example, the anti-PD-L1 antibody may be selected from the group consisting of Durvalumab (druvalumab), Atezolizumab (atezolizumab), and avelumab (avelumab).

In the present application, the anti-PD-1 antibody may comprise the HCDR3 of an antibody selected from the group consisting of Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise the HCDR2 of an antibody selected from the group consisting of: Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise HCDR1 of an antibody selected from the group consisting of Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise LCDR3 of an antibody selected from the group consisting of: Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise LCDR2 of an antibody selected from the group consisting of: Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise LCDR1 of an antibody selected from the group consisting of: Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise the VH of an antibody selected from the group consisting of Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-1 antibody may comprise the VL of an antibody selected from the group consisting of: Nivolumab (nivolumab), Pembrolizumab (pembrolizumab), Camrelizumab (camrelizumab), Toripalimab (toripalimab), sintilimab (sintilimab) and Tislelizumab (tislelizumab).

In the present application, the anti-PD-L1 antibody may comprise the 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 the 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 (druvalumab), 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 (durvalumab), 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 (durvalumab), 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, Atezolizumab, and 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:46.

In the present application, the anti-PD-1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:45.

In the present application, the anti-PD-1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:44.

In the present application, the anti-PD-1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 46; the HCDR2 comprises SEQ ID NO: 46 The amino acid sequence shown in ID NO: 45; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 44. For example, the anti-PD-1 antibody can include pembrolizumab or an antibody having the same HCDR3 (eg, having the same HCDR1-3).

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:50.

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:49.

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:48.

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:47.

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: 49; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:48; and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:47. For example, the anti-PD-1 antibody can include pembrolizumab or an antibody that has the same LCDR3 (eg, has the same LCDR1-3).

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:51.

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 can comprise the amino acid sequence set forth in SEQ ID NO:44; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:45; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:46; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:47; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:48; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:49. For example, the anti-PD-1 antibody can include pembrolizumab or an antigen binding protein having the same HCDR3 (eg, having the same HCDR1-3) and LCDR3 (eg, having the same LCDR1-3).

For example, the heavy chain variable region of the anti-PD-1 antibody may comprise the amino acid sequence set forth in SEQ ID NO:50. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same heavy chain variable region. For example, the light chain variable region of the anti-PD-1 antibody may comprise the amino acid sequence shown in SEQ ID NO:51. For example, the anti-PD-1 antibody can include pembrolizumab or an antigen binding protein having the same light chain variable region. For example, the heavy chain of the anti-PD-1 antibody may comprise the amino acid sequence set forth in SEQ ID NO:52. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same heavy chain. For example, the light chain of the anti-PD-1 antibody may comprise the amino acid sequence set forth in SEQ ID NO:53. For example, the anti-PD-1 antibody may comprise pembrolizumab or an antigen binding protein having the same light chain.

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:60.

In the present application, the anti-PD-L1 antibody comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:59.

In the present application, the anti-PD-L1 antibody comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:58.

In the present application, the anti-PD-L1 antibody comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 60; the HCDR2 comprises SEQ ID NO: 60 The amino acid sequence shown in ID NO:59; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:58. For example, the anti-PD-L1 antibody can include atezolizumab or an antibody having the same HCDR3 (eg, having the same HCDR1-3).

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:54.

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:63.

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:62.

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:61.

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: 63; the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:62; and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:61. For example, the anti-PD-L1 antibody can include atezolizumab or an antibody having the same LCDR3 (eg, having the same LCDR1-3).

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:55.

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 can comprise the amino acid sequence set forth in SEQ ID NO:58; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:59; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:60; The LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:61; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:62; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:63. For example, the anti-PD-L1 antibody can include atezolizumab or an antigen binding protein having the same HCDR3 (eg, having the same HCDR1-3) and LCDR3 (eg, having the same LCDR1-3).

For example, the heavy 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 heavy chain variable region. For example, the light chain variable region of the anti-PD-L1 antibody may comprise the amino acid sequence shown in SEQ ID NO:55. For example, the anti-PD-L1 antibody may include atezolizumab or an antigen binding protein having the same light chain variable region. For example, the heavy chain of the anti-PD-L1 antibody may comprise the amino acid sequence set forth in SEQ ID NO:56. For example, the anti-PD-L1 antibody may include atezolizumab or an antigen binding protein having the same heavy chain. For example, the light chain of the anti-PD-L1 antibody may comprise the amino acid sequence shown in SEQ ID NO:57. For example, the anti-PD-L1 antibody may include atezolizumab or an antigen binding protein having the same light chain.

Kits, uses and methods

In another aspect, the present application provides a method for detecting or assaying Siglec15, which method may comprise using the isolated antigen binding protein or the polypeptide.

In the present application, the methods may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.

For example, the method may include a method for detecting the presence and/or amount of Siglec15 for non-diagnostic purposes, which may include the steps of:

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

2) Detecting the presence and/or content of the antigen binding protein bound to the sample to determine the presence and/or expression level of Siglec15 in the sample obtained from the subject.

On the other hand, the present application provides a kit for Siglec15, which can include the use of the isolated antigen-binding protein or the polypeptide.

In the present application, the kit may further comprise instructions for use describing the method for detecting the presence and/or content of Siglec15. For example, the methods may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.

In another aspect, the present application provides the use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit, which can be used for a method for detecting the presence and/or content of Siglec15. For example, the methods may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.

In another aspect, the application provides a method of modulating an immune response, comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate, The isolated nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, and/or the pharmaceutically acceptable therapeutic agent.

In the present application, the method of modulating an immune response may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.

In another aspect, the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, and the pharmaceutical composition for prevention and alleviation and/or treatment of a disease or disorder.

On the other hand, in the present application, the kit and/or the pharmaceutical combination is used to prevent, alleviate and/or treat a disease or disorder.

For example, the disease or disorder may include abnormal bone metabolism. For example, the abnormal bone metabolism can include osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or bone metastases from cancer, osteopenia, dental Tooth loss due to periarthritis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta. For example, the abnormal bone metabolism can include osteoporosis. For example, the antigen binding proteins of the present application can be administered alone or in combination with at least one other therapeutic agent for bone-related diseases. For example, the antigen binding proteins of the present application can be administered in combination with a therapeutically effective amount of a therapeutic agent for abnormal bone metabolism. Therapeutic agents that can be administered in combination with the antigen binding proteins of the present application include, but are not limited to: bisphosphonates (eg, alendronate, etidronate, ibandronate, icardronate, paclitaxel diphosphate, risedronate and zoledronate), active vitamin D3, calcitonin and its derivatives, hormones such as estradiol, SERM (selective estrogen receptor modulator), ippro Flavonoids, vitamin K2 (menadione), calcium preparations, PTH (parathyroid hormone), NSAIDs (eg, celecoxib and rofecoxib), soluble TNF receptors (eg, , etanercept), anti-TNF-α antibody or functional fragment of said antibody (eg, infliximab), anti-PTHrP (parathyroid hormone-related protein) antibody or functional fragment of said antibody, IL-1 receptor antagonist (eg, anakinra), anti-IL-6 receptor antibody or functional fragment of said antibody (eg, tocilizumab), anti-RANKL antibody, or a functional fragment of said antibody Functional fragments (eg, denosumab) and OCIF (osteoclastogenesis inhibitory factor). Depending on the state of the abnormal bone metabolism or the desired degree of treatment and/or prevention, two, three or more drug types may be administered.

For example, the disease or disorder can include a tumor. For example, the tumor can include a solid tumor. For example, the tumor can include a tumor associated with the expression of Siglec15. The term "tumor associated with the expression of Siglec15" generally refers to tumors in which altered expression of Siglec15 leads to disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of Siglec15" may be a tumor formed by up-regulation of the expression of Siglec15 leading to disease progression or evasion of immune surveillance. The tumor associated with protein expression of Siglec15 may be a Siglec15 positive tumor. In Siglec15-positive tumors, the protein expression of Siglec15 on the tumor cell surface or in the tumor microenvironment was approximately 1%, 5%, 10%, 15%, 20%, 25%, 30% higher than normal cells, 35%, 40%, 50%, 60%, 70%, 80% or higher.

For example, the tumor can include colon cancer.

In another aspect, the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and /or the use of the pharmaceutical composition in the preparation of a medicament for preventing, relieving and/or treating a disease or condition.

In another aspect, the present application provides the use of a pharmaceutical combination in the preparation of a medicament for preventing, alleviating and/or treating a disease or condition.

For example, the disease or disorder may include abnormal bone metabolism. For example, the abnormal bone metabolism may include osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or bone metastases from cancer, osteopenia, dental Tooth loss due to periarthritis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta. For example, the abnormal bone metabolism can include osteoporosis. For example, the antigen binding proteins of the present application can be administered alone or in combination with at least one other therapeutic agent for bone-related diseases. For example, the antigen binding proteins of the present application can be administered in combination with a therapeutically effective amount of a therapeutic agent for abnormal bone metabolism. Therapeutic agents that can be administered in combination with the antigen binding proteins of the present application include, but are not limited to: bisphosphonates (eg, alendronate, etidronate, ibandronate, icadronate, paclitaxel diphosphate, risedronate and zoledronate), active vitamin D3, calcitonin and its derivatives, hormones such as estradiol, SERM (selective estrogen receptor modulator), ippro Flavonoids, vitamin K2 (menadione), calcium preparations, PTH (parathyroid hormone), NSAIDs (eg, celecoxib and rofecoxib), soluble TNF receptors (eg, , etanercept), anti-TNF-α antibody or functional fragment of said antibody (eg, infliximab), anti-PTHrP (parathyroid hormone-related protein) antibody or functional fragment of said antibody, IL-1 receptor antagonist (eg, anakinra), anti-IL-6 receptor antibody or functional fragment of said antibody (eg, tocilizumab), anti-RANKL antibody, or a functional fragment of said antibody Functional fragments (eg, denosumab) and OCIF (osteoclastogenesis inhibitory factor). Depending on the state of the abnormal bone metabolism or the desired degree of treatment and/or prevention, two, three or more drug types may be administered.

For example, the tumor can include colon cancer.

In another aspect, the present application provides a method of preventing and/or treating a disease or disorder, comprising administering to a subject in need the isolated antigen-binding protein, the isolated nucleic acid molecule, the The carrier, the cell, and the pharmaceutical composition.

In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering the pharmaceutical combination to a subject in need thereof.

For example, the disease or disorder may include abnormal bone metabolism. For example, the abnormal bone metabolism may include osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone destruction associated with multiple myeloma or bone metastases from cancer, osteopenia, dental Tooth loss due to periarthritis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta. For example, the abnormal bone metabolism can include osteoporosis. For example, the antigen binding proteins of the present application can be administered alone or in combination with at least one other therapeutic agent for bone-related diseases. For example, the antigen binding proteins of the present application can be administered in combination with a therapeutically effective amount of a therapeutic agent for abnormal bone metabolism. Therapeutic agents that can be administered in combination with the antigen binding proteins of the present application include, but are not limited to: bisphosphonates (eg, alendronate, etidronate, ibandronate, icardronate, paclitaxel diphosphate, risedronate and zoledronate), active vitamin D3, calcitonin and its derivatives, hormones such as estradiol, SERM (selective estrogen receptor modulator), ippro Flavonoids, vitamin K2 (menadione), calcium preparations, PTH (parathyroid hormone), NSAIDs (eg, celecoxib and rofecoxib), soluble TNF receptors (eg, , etanercept), anti-TNF-α antibody or functional fragment of said antibody (eg, infliximab), anti-PTHrP (parathyroid hormone-related protein) antibody or functional fragment of said antibody, IL-1 receptor antagonist (eg, anakinra), anti-IL-6 receptor antibody or functional fragment of said antibody (eg, tocilizumab), anti-RANKL antibody, or a functional fragment of said antibody Functional fragments (eg, denosumab) and OCIF (osteoclastogenesis inhibitory factor). Depending on the state of the abnormal bone metabolism or the desired degree of treatment and/or prevention, two, three or more drug types may be administered.

For example, the disease or disorder can include a tumor. For example, the tumor can include a solid tumor. For example, the tumor can include a tumor associated with the expression of Siglec15. The term "tumor associated with the expression of Siglec15" generally refers to tumors in which altered expression of Siglec15 leads to disease progression or evasion of immune surveillance. For example, the "tumor associated with the expression of Siglec15" may be a tumor formed by up-regulation of the expression of Siglec15 leading to disease progression or evasion of immune surveillance. The tumor associated with protein expression of Siglec15 may be a Siglec15 positive tumor. In Siglec15-positive tumors, the protein expression of Siglec15 on the tumor cell surface or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30% higher than normal cells, 35%, 40%, 50%, 60%, 70%, 80% or higher.

For example, the tumor can include colon cancer.

The pharmaceutical compositions, combinations, and methods described herein can be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like. The pharmaceutical compositions and methods described in this application can be used for other disease conditions that depend on an immune response, such as inflammation, immune diseases, and infectious diseases.

In the present application, the subject may include a human or a non-human animal. For example, the non-human animal may be selected from the group consisting of monkey, chicken, goose, cat, dog, mouse and rat. In addition, non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals. The person may be Caucasian, African, Asian, Semitic, or other race, or a hybrid of various races. As another example, the human can be an elderly, adult, adolescent, child, or infant.

The effective amount in humans can be extrapolated from the effective amount in experimental animals. For example, Freireich et al. describe the correlation of doses (based on milligrams per square meter of body surface) in animals and humans (Freireich et al., Cancer Chemother. Rep. 50, 219 (1966)). Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).

Not to be limited by any theory, the following examples are only intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Immune antigen activity verification

1.1 Antigen coating: prepare a solution of husiglec-15-hFc antigen (human Siglec-15Fc, SinoBiological, Cat. No. 13976-H02H) diluted in concentration gradient with DPBS solution, and coat the 96-well plate overnight at 4°C.

1.2 Washing: Take the overnight coated 96-well plate and wash it three times with PBST solution.

1.3 Blocking: The antigens were blocked with DPBS (containing 3% BSA), placed at room temperature, and blocked for 1 hour.

1.4 Ligand binding: Siglec-15 antibody (mouse-human chimeric antibody (5G12, human IgG1), Acrobiosystem) was quantitatively diluted with DPBS (containing 1% BSA) solution, and coated for 1 hour at 37°C.

1.5 Secondary antibody binding: discard the reaction solution, wash three times with PBST solution, add secondary antibody (HRP-goat anti-human IgG, F(ab')2 secondary antibody, source: Jackson, catalog number: 109-035-006), each 100 μl was added to the well, and the reaction was placed at 37°C for 1 hour.

1.6 ELISA detection: Discard the reaction solution, wash it three times with PBST solution; add TMB solution and H ₂ SO ₄ stop solution successively, and after slight shaking turns yellow, detect the OD value at 450 nm.

As shown in Figure 1, the results of the husiglec-15-hFc antigen activity test by ELISA showed that the husiglec-15-hFc antigen has good antigenic activity and can be used as a mouse immunogen in the later hybridoma experiments.

Example 2 Screening antigen activity verification

2.1 Antigen coating: prepare a concentration gradient dilution of husiglec-15-his antigen (human Siglec-15his, Acrobiosystem, product number: SG5-H52H3) solution with DPBS solution, cyno-siglec-15-hFc (cynomolgus monkey Siglec-15his, Acrobiosystem, product number: SG5-C52H6) took a 96-well plate, added 100 μl to each well, and coated overnight at 4°C.

Subsequent experiments are the same as steps 2-6 of case 1.

As shown in Figure 2A, the results of cyno-siglec-15-his antigen activity determined by ELISA showed that it has good antigenic activity and can be used for screening antigens in the supernatant of hybridoma cells in the later stage. As shown in Figure 2B, the husiglec determined by ELISA The results of -15-his antigenic activity show that it has good antigenic activity and can be used for the detection of antibody titer in the serum of immunized mice.

Example 3 Preparation and screening of anti-human Siglec-15 monoclonal antibody

Anti-human Siglec-15 monoclonal antibody was produced by immunizing mice, and the experiment used SJL white mice, female, 6 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). Breeding environment: SPF grade. After the mice were purchased, the mice that had been adapted to the environment were immunized with recombinant protein husiglec-15-Fc (human siglec-15Fc, SinoBiological, product number: 13976-H02H) (50ug) and RIBI adjuvant after being reared in a laboratory environment for 1 week. . After 4-5 immunizations, serum antibody levels were monitored during the period, and mice with high antibody titers in serum were selected for booster immunization. The mice were sacrificed 3 days later, and spleen cells were collected and fused with myeloma cells. Hybridoma cells were obtained by fusing spleen lymphocytes with myeloma cells Sp2/0 cells (ATCC, catalog number: BDXB-0001) using an optimized electrofusion procedure.

As shown in Table 1, experimental animals and immunization information.

Table 1 Test animals and immunization information

小鼠类型mouse type	SJL鼠(北京维通利华实验动物技术有限公司)SJL rat (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.)
免疫抗原immune antigen	husiglec-15-Fc抗原husiglec-15-Fc antigen
免疫方法immunization method	初次免疫50ug/只，后续25μg/只，腹腔注射(ip)Initial immunization 50ug/a, follow-up 25μg/a, intraperitoneal injection (ip)
免疫次数 Immunization times	55
最后加强免疫final booster	50μg husiglec-15-Fc蛋白，腹腔注射(ip)，在融合前三天进行50 μg husiglec-15-Fc protein, intraperitoneally (ip), three days before fusion

3.1 Detection of serum titer in mice

3.1.1 Antigen coating: Prepare 1 μg/ml husiglec-15-his antigen (human Siglec-15his, Acrobiosystem, Cat. No.: SG5-H52H3) solution in DPBS solution, and coat the 96-well plate overnight at 4°C.

3.1.2 Washing: Take overnight-coated 96-well plate and wash three times with PBST solution.

3.1.3 Blocking: The antigens were blocked with DPBS (containing 3% BSA), placed at room temperature, and blocked for 1 hour.

3.1.4 Antibody binding: the serum solution was diluted with DPBS (containing 1% BSA) solution, and the negative control was serum from unimmunized mice. Positive control wells were Siglec-15 antibody (mouse-human chimeric antibody (5G12, Human IgG1), Acrobiosystem). It was placed at room temperature and reacted for 1 hour.

3.1.5 Secondary antibody binding: discard the reaction solution, wash three times with PBST solution; add secondary antibody (goat anti-mouse IgG F(ab')2 secondary antibody, coupled to HRP, source: invitrogen, product number: 31436), positive control A secondary antibody (HRP-goat anti-human IgG, F(ab')2 secondary antibody, source: Jackson, catalog number: 109-035-006) was added to the well and allowed to react at room temperature for 1 hour.

3.1.6 ELISA detection: Discard the reaction solution, wash with PBST solution three times; add TMB solution and H ₂ SO ₄ stop solution successively, after slight shaking, the OD value is detected at 450 nm.

As shown in Figure 3, serum titers of multiple immunized mice. The experimental results show that among the 5 immunized mice, the 2#, 3#, and 4# mice have higher efficacy values, all reaching 437.4k, reaching the titer that can be fused, and subsequent immunization and fusion experiments can be arranged.

As shown in Table 2, the serum potency values of multiple immunized mice.

Table 2 Serum titers of immunized mice

1#1#	2#2#	3#3#	4#4#	5#5#
5.4K5.4K	437.4K437.4K	437.4K437.4K	437.4K437.4K	1.8K1.8K

3.2 Hybridoma fusion

3.2.1 Mix spleen cells and SP2/0 mouse myeloma cells at a ratio of 5:1 to 2:1, centrifuge, and co-precipitate. After removing the supernatant, resuspend the cells with an appropriate amount of fusion buffer, and adjust the mixed cell density to 1×10 ⁷ cells/ml for electrofusion. After the fusion was completed, the cells were placed in the electrode box for 5 minutes, and the cells were added to the 1×HAT selection medium in 20% FBS, transferred to a 96-well cell culture plate, and cultured in a 37°C, 5% CO ₂ incubator. . Change the fresh medium once every 7-8 days.

3.2.2 After the 10th day of culture (or longer, depending on the cell growth state), take the supernatant, measure the antibody expression in the hybridoma supernatant by FACS and ELISA, and screen out the hybridoma expressing the specific anti-Siglec-15 antibody cell. The positive clones obtained by the primary screening were transferred to 24-well plates for culture, and then the supernatant was identified by rescreening, and the positive clones were selected for 1-2 rounds of subcloning, and the hybridoma monoclonal cells stably expressing the specific anti-Siglec-15 antibody were screened again. , and select the hybridoma cell lines with the highest expression, cryopreserved cell samples and Trizol samples.

3.3 Antigen-binding protein sequencing

The present application utilizes molecular biology techniques to obtain the antigen-binding protein sequence in Siglec-15 positive hybridoma cells, and perform chimeric and humanization transformation.

3.4 Humanized design of anti-human Siglec-15 antibody

In order to reduce the immunogenicity of murine antibodies, the selected biologically active antibodies can optionally be humanized. Humanization of murine monoclonal antibodies is carried out according to methods disclosed in many literatures in the art. Briefly, CDR grafting can be performed using human antibody constant domains in place of parental (murine antibody) constant domains, selecting human germline antibody sequences based on the homology of the murine and human antibodies. Then, based on the three-dimensional structure of the murine antibody, back-mutation of the amino acid residues of VL and VH can be performed to replace the constant region of the murine antibody with the human constant region to obtain the final humanized binding protein.

As shown in Table 3, Table 4 and Table 5, the amino acid sequences of CDR, heavy chain variable region VH, light chain variable region VL, FR, light chain constant region CL and heavy chain constant region CH of the antigen binding protein of the present application .

Table 3 Sequence information of CDR, heavy chain variable region VH, and light chain variable region VL of the antigen binding protein of the present application

Table 4 Sequence information of the FRs of the antigen-binding proteins of the present application

Table 5 Sequence information of the light chain constant region CL and the heavy chain constant region CH of the antigen-binding protein of the present application

轻链恒定区light chain constant region	SEQ ID NO:43SEQ ID NO: 43
重链恒定区IgG1Heavy chain constant region IgG1	SEQ ID NO:39SEQ ID NO: 39
重链恒定区IgG2heavy chain constant region IgG2	SEQ ID NO:40SEQ ID NO: 40
重链恒定区IgG3Heavy chain constant region IgG3	SEQ ID NO:41SEQ ID NO: 41

Heavy chain constant region IgG4

SEQ ID NO: 42

3.5 Expression and purification of antibody proteins

A plasmid was constructed according to the antigen-binding protein sequence of the present application, transiently expressed in Expi293 cells (Thermo Fisher, Cat. No. A14527CN), and the antibody expression plasmid was extracted using a large-scale extraction kit. Solution 1: Dilute 15 μg of plasmid with 1 ml of culture medium and mix well. Solution 2: Dilute 60 μl of transfection reagent with 1 ml of culture medium and mix well. Add solution 2 to solution 1, mix well, 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 expression for one week, collect the supernatant, 8000 rpm /min centrifugation for 5 minutes.

Antibody protein purification by Protein A affinity chromatography column:

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

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

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

(4) Elution: citrate 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 280 nm was read using a NanoDrop instrument.

(5) Dialysis: suck the high-concentration protein into a dialysis bag and place it in a beaker of 1×PBS for dialysis. The purity test using high performance liquid chromatography LC-20AT and gel chromatography column is qualified, and the endotoxin test is qualified.

Example 4 Binding of chimeric Siglec15 antigen-binding protein to human Siglec15 expressed on cells

The binding ability of Siglec15 antigen-binding protein to human Siglec15 expressed on the surface of CHOK1 cells was determined based on a flow cytometry assay. The binding capacity of different Siglec15 antigen-binding proteins was determined by comparing their binding curves to human Siglec15 expressed on the surface of CHOK1 cells.

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

4.2 Prepare a positive control antibody (SPH-Sg-PC-1, Taizhou Baiying Biotechnology Co., Ltd., item number B218501 with PBS containing 2% FBS respectively, heavy chain variable region sequence as shown in SEQ ID NO: 37, light The chain variable region sequence is shown in SEQ ID NO: 38), the negative control antibody (human IgG1, novoprotein, product number NC002) and the maximum concentration of the antigen-binding protein of the present application is 15 μg/ml, 5-fold dilution, 8 points, well diluted The samples were added to a 96-well plate and incubated at 4 degrees Celsius for 1 hour.

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

4.4 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 degrees Celsius for 0.5 hours.

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

4.6 The cells were resuspended with 2% FBS in PBS, and the median fluorescence value (MFI) of the PE channel was measured by flow cytometry.

The above experiments show that the chimeric Siglec15 antigen-binding protein of the present application has the binding activity to the human Siglec15 overexpressed on CHOK1 cells, and is superior to the positive antibody.

As shown in FIG. 4 , the binding curve of the chimeric Siglec15 antigen-binding protein to human Siglec15 expressed on the surface of CHOK1 cells was determined based on flow cytometry.

As shown in Table 6, the EC ₅₀ value and the highest MFI value of the binding of the chimeric Siglec15 antigen-binding protein to human Siglec15 expressed on the surface of CHOK1 cells were determined based on flow cytometry.

Table 6 EC ₅₀ value and highest MFI value of chimeric Siglec15 antigen-binding protein binding to Siglec15 expressed on the surface of CHOK1 cells

编号Numbering	EC ₅₀(μg/ml) _EC50 (μg/ml)	最高MFI值Highest MFI value
阳性对照抗体positive control antibody	0.29560.2956	39462.2639462.26
阴性对照抗体Negative control antibody	～~	243.61243.61
Ab0Ab0	0.070460.07046	41452.9841452.98

Example 5 Biological activity of chimeric Siglec15 antigen-binding protein

Siglec15 is a member of the Siglecs family expressed on the surface of immune system cells. In the tumor microenvironment, it can inhibit the activation and proliferation of T cells, thereby inhibiting the antitumor activity of T cells. After the Siglec15 antigen-binding protein binds to Siglec15, it can block its inhibitory effect on T cells, enhance the activation and proliferation of T cells, and then increase the expression and secretion of IFN-γ.

In the presence of Siglec15, the Siglec15 antigen-binding protein was co-incubated with PBMCs extracted from human blood for a certain period of time. Finally, Siglec15 was determined by detecting the proliferation of CD4 and CD8 T cells in the co-incubated PBMCs and the expression of IFN-γ in the supernatant of the medium. Whether antigen-binding protein can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the blood of subjects.

5.1 Detection of CD4 and CD8 T cell proliferation

5.1.1 Add 100 μl of OKT3 (novoprotein, Cat. No. GMP-A018) to a 96-well cell plate (Corning, 3599), and incubate at 4°C overnight. After coating, wash three times with PBS and set aside.

5.1.2 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 final concentration of Siglec15 antigen to 5 μg/ml, and add the diluted sample to a 96-well plate. Incubate the cell plate at 37°C for 30min.

5.1.3 The cryopreserved human peripheral mononuclear cells (PBMC) were thawed in a 37° C. water bath, resuspended in PBS, centrifuged at 400 g for 5 min, and the supernatant was discarded. Resuspend cells in PBS to adjust cell density, add 0.5 μM CFSE (Invitrogen, Cat. No. 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, 400 g, 5 minutes Centrifuge and discard the supernatant.

5.1.4 Wash cells with PBS three times. After resuspending PBMCs in 1640 medium with serum, they were added to the incubated antibody wells and incubated at 37°C for 64h. After the incubation, the cell supernatant was collected for use.

5.1.5 Transfer the PBMC in the cell reaction plate to a 96-well U-shaped plate (Corning, Cat. No. 3799) with FACS buffer (PBS+2% FBS), centrifuge at 400 g for 5 min and discard the supernatant. APC anti-human CD8 (BioLegend, 344722) and PE-Cy7 anti-human CD4 (BioLegend, 357410) antibodies were diluted with FACS buffer according to the instructions, 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.

5.1.6 Repeat washing of cells 3 times with FACS buffer. The 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.

5.2 Use human IFN-g Elisa Kit (RnD, catalog number DY285B) to detect the expression of IFN-γ in the supernatant of the medium

5.2.1 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.

5.2.2 After coating, use PBST (0.05%

20in PBS, pH 7.2-7.4) washed three times, after washing the plate, put the 96-well plate upside down on a clean laboratory paper towel, tap several times to remove the residual liquid in the well.

5.2.3 Block with PBST containing 1% BSA, 300 μl/well, block at room temperature for 2 hours. After blocking, wash three times with PBST.

5.2.4 Dilute the samples and standards with PBST containing 1% BSA to the working concentration, add 100 μl/well of samples and standards into a 96-well plate, cover the plate after completion, seal the gap at the interface, and incubate at room temperature for 2h. After incubation, wash three times with PBST.

5.2.5 Dilute the detection antibody to 200ng/ml with PBST containing 1% BSA, add 100μl/well of the diluted detection antibody to the 96-well plate, cover the plate, seal the gap at the interface, and incubate at room temperature for 2h. After incubation, wash three times with PBST.

5.2.6 Dilute streptavidin-HRP to the working concentration at a ratio of 1:40 with PBST containing 1% BSA, then add it to a 96-well plate, 100 μl/well, and incubate at 37°C for 20 min (during dilution and incubation) Be careful to avoid light). After incubation, wash three times with PBST.

5.2.7 Add 100 μl/well of the TMB mixture to a 96-well plate, and incubate at 37°C for 15-20 minutes (this step needs to be protected from light).

5.2.8 After the reaction, add 50 μl of stop solution to the reaction, and tap the 96-well plate to mix well. Immediately after terminating the reaction, read the absorbance at 450 nm of the 96-well plate with a microplate reader. The level of INF-γ in the samples was calculated from the standard.

The above experiments show that the chimeric 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.

As shown in Figures 5A, 5B, and 5C, the chimeric Siglec15 antigen-binding protein can promote the proliferation of CD8 and CD4 T cells and the expression of IFN-γ in the supernatant of the medium, and its activity is better than that of the positive antibody.

As shown in Table 7, the chimeric Siglec15 antigen-binding protein can block the proliferation-inhibitory ability of Siglec15 on PBMCs in the blood of subjects.

Table 7 The ability of chimeric Siglec15 to bind to block Siglec15 to inhibit the proliferation of PBMCs in the blood of subjects

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

The binding ability of Siglec15 antigen-binding protein to human Siglec15 expressed on the surface of CHOK1 cells was determined based on a flow cytometry assay. By comparing the binding curves of different humanized Siglec15 antigen-binding proteins of the present application with human Siglec15 expressed on the surface of CHOK1 cells, the binding capacity was determined.

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

6.2 Prepare positive control antibody (SPH-Sg-PC-1, Taizhou Baiying Biotechnology Co., Ltd., Item No. B218501), negative control antibody (human IgG1, novoprotein, Item No. NC002) and this application with PBS containing 2% FBS The maximum concentration of antigen-binding protein antibody was 15 μg/ml, 5-fold dilution, 8 points, the diluted samples were added to 96-well plates, and incubated at 4 degrees Celsius for 1 hour.

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

6.4 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 degrees Celsius for 0.5 hours.

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

6.6 The cells were resuspended with 2% FBS in PBS, and the median fluorescence intensity (MFI) of the PE channel was measured by flow cytometry.

The above experiments show that the humanized Siglec15 antigen-binding protein of the present application has the binding activity to the human Siglec15 overexpressed on CHOK1 cells, and is superior to the positive antibody.

As shown in FIG. 6A and FIG. 6B , the binding curve of humanized Siglec15 antigen-binding protein to human Siglec15 expressed on the surface of CHOK1 cells was determined based on flow cytometry.

As shown in Table 8, the _EC50 value and the highest MFI value of the humanized Siglec15 antigen-binding protein binding to human Siglec15 expressed on the surface of CHOK1 cells were determined based on flow cytometry.

Table 8 EC ₅₀ value and highest MFI value of humanized Siglec15 antigen-binding protein binding to human Siglec15 expressed on the surface of CHOK1 cells

编号Numbering	EC ₅₀(μg/ml) _EC50 (μg/ml)	最高MFI值Highest MFI value
阳性对照抗体positive control antibody	0.05990.0599	27625.1327625.13
阴性对照抗体Negative control antibody	～~	288.74288.74
Ab0Ab0	0.03340.0334	24080.7724080.77
Ab1Ab1	0.08040.0804	25061.8925061.89
Ab2Ab2	0.03800.0380	23658.7223658.72
Ab3Ab3	0.04670.0467	23381.6823381.68
Ab4Ab4	0.07230.0723	26642.7926642.79
Ab5Ab5	0.05600.0560	26104.6326104.63
Ab6Ab6	0.03450.0345	24640.9724640.97
Ab7Ab7	0.07180.0718	25183.2925183.29
Ab8Ab8	0.04600.0460	24927.3924927.39
Ab9Ab9	0.06090.0609	23493.6823493.68

Example 7 Biological activity of humanized 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 at 4°C overnight. After coating, wash three times with PBS and set aside.

7.1.2 Dilute the antibody to be tested 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. Incubate the cell plate at 37°C for 30min.

7.1.3 Frozen human peripheral mononuclear cells (PBMC) were thawed in a 37°C water bath, resuspended in PBS, centrifuged at 400 g for 5 min, and the supernatant was discarded. Resuspend cells in PBS to adjust cell density, add 0.5 μM CFSE (Invitrogen, Cat. No. 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, 400 g, 5 minutes Centrifuge and discard the supernatant.

7.1.4 Wash cells three times with PBS. After resuspending PBMCs in 1640 medium with serum, they were added to the incubated antibody wells and incubated at 37°C for 64h. After the incubation, the cell supernatant was collected for use.

7.1.5 Transfer the PBMC in the cell reaction plate to a 96-well U-shaped plate (Corning, Cat. No. 3799) with FACS buffer (PBS+2% FBS), centrifuge at 400 g for 5 min and discard the supernatant. APC anti-human CD8 (BioLegend, 344722) and PE-Cy7 anti-human CD4 (BioLegend, 357410) antibodies were diluted with FACS buffer according to the instructions, 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 Repeat washing of cells 3 times with FACS buffer. The 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 supernatant of the medium

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 After coating, use PBST (0.05%

7.2.3 Block with PBST containing 1% BSA, 300 μl/well, block at room temperature for 2 hours. After blocking, wash three times with PBST.

7.2.4 Dilute the samples and standards with PBST containing 1% BSA to the working concentration, add 100 μl/well of the samples and standards into the 96-well plate, cover the plate after completion, seal the gap at the interface, and incubate at room temperature for 2h. After incubation, wash three times with PBST.

7.2.5 Dilute the detection antibody to 200ng/ml with PBST containing 1% BSA, add 100μl/well of the diluted detection antibody to the 96-well plate, cover the plate, seal the gap at the interface, and incubate at room temperature for 2h. After incubation, wash three times with PBST.

7.2.6 Dilute streptavidin-HRP to the working concentration at a ratio of 1:40 with PBST containing 1% BSA, then add it to a 96-well plate, 100 μl/well, and incubate at 37°C for 20 min (during 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 to 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, add 50 μl of stop solution to the reaction, and tap the 96-well plate to mix well. Immediately after terminating the reaction, read the absorbance at 450 nm of the 96-well plate with a microplate reader. The level of INF-γ in the samples was calculated from the standard.

The above experiments show that the Siglec15 antigen-binding protein of the applicant can block the proliferation-inhibitory ability of Siglec15 on PBMCs in the blood of subjects.

As shown in Figure 7A, 7B, 7C, the humanized Siglec15 antigen binding protein can promote the proliferation of CD8 and CD4 T cells and promote the expression of IFN-γ in the supernatant of the medium, and the activity is better than that of the positive antibody.

As shown in Table 9, the Siglec15 antigen-binding protein can block the ability of Siglec15 to inhibit the proliferation of PBMCs in the blood of subjects.

Table 9 Siglec15 antigen-binding protein blocks the ability of Siglec15 to inhibit the proliferation of PBMCs in the blood of subjects

Example 8 Binding affinity of Siglec15 antigen-binding protein

Biacore was used to detect the binding affinity of different Siglec15 antigen-binding proteins to antigen (Siglec15 protein, human, recombinant (ECD, His Tag), source: Acro, Cat. No.: SG5-C5253) protein.

Reagent preparation. Running reagents: containing 10 mM N-(2-hydroxyethyl)piperazine-N-2 sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20 (Tween-20), pH adjusted to 7.4; Human IgG(Fc) Capture Kit (Cat. No. BR-1008-39, GE), including: mouse anti-human IgG(Fc) antibody (0.5mg/mL), immobilized Reagents (10mM Sodium Acetate, pH5.0), Regeneration Reagent (3M Magnesium Chloride); Amino Coupling Kit (Cat. No. BR100050, GE), including: 115mg N-Hydroxysuccinimide (NHS), 750mg 1- Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10.5 mL of 1 M ethanolamine (pH 8.5). Add 10 mL of deionized water to each tube of EDC and NHS, respectively, and store in aliquots at -18°C to a lower temperature. The shelf life is two months. (Refer to GE Amino Coupling Instruction Manual "22-0510-62AG").

Chip preparation. The mouse anti-human IgG (Fc) antibody was diluted to 25 μg/mL with immobilization reagent (10mM sodium acetate, pH 5.0), about 100 μL of mouse anti-human IgG (Fc) antibody was used for each channel of the chip, and about 1900 μL was used for immobilizing eight channels. 100 μL of mouse anti-human IgG (Fc) antibody was added to the immobilization reagent. First, the surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL/min for 420 s. Next, 25 μg/mL of mouse anti-human IgG (Fc) antibody was injected into the experimental channel (FC2) at a flow rate of 10 μL/min for about 420 s, and the fixed amount was about 9000 to 14000 RU. Finally, the chip was blocked with 1 M ethanolamine at 10 μL/min for 420 s. The reference channel (FC1) performs the same operation as the test channel (FC2). (Refer to GE Human IgG(Fc) Capture Kit Instruction Manual "22-0648-88AD").

capture ligand. Eleven Siglec15 antigen-binding proteins were diluted to 5 μg/mL with running reagent, and injected into the experimental channel (FC2) at a flow rate of 10 μL/min, and captured for 40 s. The reference channel (FC1) did not require ligand capture.

Analyte single concentration analysis. The Siglec15 antigen was diluted to 400 nM with running reagent. After capturing the Siglec15 antigen-binding protein, the diluted Siglec15 antigen was sequentially combined at a flow rate of 30 μL/min for 90s, dissociated for 210s, and injected into the experimental channel and the reference channel. At the same time, each antibody was set to a 0 concentration. After each Siglec15 antigen-binding protein analysis, the chip needs to be regenerated with 3M magnesium chloride at a flow rate of 20 μL/min for 30 s to wash away ligands and undissociated analytes.

Analyte single concentration analysis. Antigen was diluted to 200 nM with running reagent. After capturing the antibody, the diluted antigens were sequentially combined for 90 seconds and dissociated for 210 seconds at a flow rate of 30 μl/min, and then injected into the experimental channel and the reference channel, and each antigen-binding protein was set to a concentration of 0. After each antigen-binding protein analysis, the chip was regenerated with 3M magnesium chloride at a flow rate of 20 μl/min for 30 seconds to wash away ligand as well as undissociated analyte.

All operation steps are carried out in the running reagent, and the analytical reagents of SPR need to be filtered and degassed. KD values for each antibody were calculated using _Biacore8K analysis software. The reference channel (FC1) is used for background subtraction.

The above experiments show that the Siglec15 antigen-binding protein of the present applicant has binding affinity to the antigen.

As shown in Table 10, the binding affinity of different humanized Siglec15 antigen-binding proteins to antigen proteins was detected by Biacore.

Table 10 Binding affinity of Siglec15 antigen-binding protein to antigenic protein

编号Numbering	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	2.89E+052.89E+05	5.96E-045.96E-04	2.06E-092.06E-09
Ab0Ab0	1.14E+051.14E+05	4.41E-044.41E-04	3.86E-093.86E-09
Ab1Ab1	1.27E+051.27E+05	4.49E-044.49E-04	3.54E-093.54E-09
Ab2Ab2	1.14E+051.14E+05	3.81E-043.81E-04	3.34E-093.34E-09
Ab3Ab3	1.04E+051.04E+05	4.13E-044.13E-04	3.95E-093.95E-09
Ab4Ab4	1.39E+051.39E+05	4.54E-044.54E-04	3.27E-093.27E-09
Ab5Ab5	1.14E+051.14E+05	4.15E-044.15E-04	3.66E-093.66E-09
Ab6Ab6	1.17E+051.17E+05	4.12E-044.12E-04	3.52E-093.52E-09
Ab7Ab7	1.31E+051.31E+05	4.33E-044.33E-04	3.31E-093.31E-09

Ab8Ab8	1.48E+051.48E+05	3.93E-043.93E-04	2.65E-092.65E-09
Ab9Ab9	1.21E+051.21E+05	4.80E-044.80E-04	3.97E-093.97E-09

Example 9 Species cross-reactivity test of Siglec15 antigen-binding protein

In this test, Siglec15 antigen binding protein was detected with human Siglec15-his (Acro, cat. No. 5G5-H52H3), cynomolgus monkey Siglec15-hFc (Acro, cat. No. SG5-C5253), mouse Siglec15-his (Acro, cat. No. SG5-M52H7) protein binding.

9.1 Prepare human Siglec15-his, cynomolgus monkey Siglec15-hFc and mouse Siglec15-his 1ug/ml with PBS respectively, add them to a 96-well plate (Corning, Cat. No. 9018), overnight at 4 degrees.

9.2 The next day, the coating solution was discarded, and the plate was washed three times with PBS with 0.05% Tween20.

9.3 Incubate with 3% BSA in PBS at 37°C for 2 hours, and wash the plate 3 times with 0.05% Tween20 in DPBS.

9.4 Prepare the antibody to be tested with PBS containing 1% BSA to a maximum concentration of 15ug/ml, 5-fold dilution, and 8 concentration points. Add to 96-well plate and incubate for 1 hour at room temperature. Plates were washed 3 times with 0.05% Tween20 in PBS.

9.5 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 incubate at room temperature 0.5 hours. Plates were washed 3 times with 0.05% Tween20 in PBS.

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

9.7 Add 100ul of 2N H ₂ SO ₄ stop solution to each well, read the value with a microplate reader at a wavelength of 450 nm and record it.

The above experiments show that the Siglec15 antigen-binding protein of the applicant has the ability to bind to human, monkey and murine Siglec15 antigens.

As shown in Figures 8A, 8B, 8C, the humanized Siglec15 antigen binding protein has binding curves to human, monkey and murine Siglec15 antigens.

As shown in Table 11, the binding ability of the humanized Siglec15 antigen-binding proteins to human, monkey and murine Siglec15 antigens.

Table 11 Binding ability of humanized Siglec15 antigen binding protein to human, monkey and murine Siglec15 antigen

Example 10 Antitumor efficacy test of Siglec15 antigen-binding protein on wild-type C57BL/6N animal model

In this test, 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) were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. Mice were acclimated for 7 days after arrival before the study began.

Cells: Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. and routinely subcultured according to the instructions; SPH genetically modified MC38 cells to overexpress human Siglec15, and the cells were named MC38/ hSiglec15 cells were used for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in serum-free medium and adjusted for cell density. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of wild-type C57BL/6N mice subcutaneously to establish the MC38/hSiglec15 tumor-bearing mouse model.

Dosing:

On the 6th day of tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with tumor volume in the range of 100 mm ³ to 190 mm ³ were selected and grouped by tumor volume (6 mice in each group). , 9, 12, 15, 18, 21 days of administration, negative control antibody (human IgG), positive control antibody (SPH-Sg-PC-1, source: Taizhou Baiying Biotechnology Co., Ltd., product number B218501), this Antigen-binding proteins were applied, and the tumor volume and body weight changes of mice in each group were monitored during the administration period. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 24th to 26th day after inoculation. The calculation formula is as follows: TGI(%)=[1-(Ti-T0)/(Vi- V0)]×100; where Ti: the mean tumor volume in the administration group, T0: the mean tumor volume in the administration group on D0 day, Vi: the mean tumor volume in the isotype control group, V0: the tumor volume in the isotype control group on D0 day average. Determination of tumor volume: vernier calipers were used to measure the long diameter (a) and wide diameter (b) of the tumor, 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.

Dosage and mode of administration are shown in Table 12.

Table 12 Dosing trial design

编号Numbering	给药剂量Dosage	给药次数number of doses	给药方式way 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
Ab6Ab6	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection

Ab7

10mg/kg

Q3DX6*

intraperitoneal injection

*Dosed every 3 days for a total of 6 times.

As shown in Figure 9A, in the first experiment, the antitumor efficacy of the antigen-binding protein Ab0 in wild-type C57BL/6N mice was detected.

As shown in Figure 9B, the body weight changes of wild-type C57BL/6N mice after the administration of the antigen binding protein Ab0 in the first experiment.

As shown in Figure 10A, in the second experiment, the antitumor efficacy of antigen binding proteins Ab1, Ab6 and Ab7 in wild-type C57BL/6N mice was detected.

As shown in FIG. 10B , changes in body weight of wild-type C57BL/6N mice following administration of antigen binding proteins Ab1, Ab6 and Ab7 in the second experiment.

As shown in Table 13 and Table 14, in the first test, on the 26th day after inoculation, the antigen-binding protein Ab0 of the present application significantly inhibited the growth of tumors at a dose of 10 mg/kg, and the tumor volume inhibition rate was 83%; In the second experiment, on the 24th day after inoculation, the antigen binding proteins Ab1, Ab6 and Ab7 of the present application significantly inhibited tumor growth at a dose of 10 mg/kg, and the tumor volume inhibition rates were 99%, 60% and 54%, respectively.

The above experiments showed that the antigen binding proteins Ab0, Ab1, Ab6 and Ab7 of the present application significantly inhibited the growth of tumors, and had no significant effect on the body weight of wild-type C57BL/6N mice.

Table 13 The ability of the antigen-binding protein Ab0 of the present application to inhibit tumor growth in vivo on the 26th day in the first test

Note: *: Compared with negative control antibody tumor volume, P<0.05; **: Compared with negative control antibody tumor volume, P<0.01.

Table 14 Antigen binding proteins Ab1, Ab6 and Ab7 of the present application inhibit tumor growth in vivo on day 24 in the second experiment

Example 11 Antitumor efficacy test of Siglec15 antigen-binding protein on human Siglec15 transgenic mice

In this test, MC38/hSiglec15 cells are 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 C57BL/6 background human Siglec15 transgenic mice (6 weeks) were purchased from Shanghai Southern Model Biotechnology Co., Ltd. Mice were acclimated for 7 days after arrival before the study began.

Cells: Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. and routinely subcultured according to the instructions; MC38 cells were genetically modified to overexpress human-derived Siglec15, and the cell was named MC38/human Siglec15 cells for subsequent in vivo experiments. The cells were collected by centrifugation, resuspended in serum-free medium and adjusted for cell density. 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.

Dosing:

On the 6th day of tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with tumor volume in the range of 90 mm ³ to 130 mm ³ were selected and grouped by tumor volume (5 to 6 mice in each group). Administration on the 6th, 9th, 12th, 15th, 18th and 21st days, negative control antibody (human IgG), positive control antibody (SPH-Sg-PC-1, source: Taizhou Baiying Biotechnology Co., Ltd., product number B218501) The antigen-binding protein of the present application was monitored for changes in tumor volume and body weight of the mice in each group during administration, and the monitoring frequency was 2 times/week, and the monitoring was continued 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. The calculation formula is as follows: TGI(%)=[1-(Ti-T0)/(Vi-V0) ]×100; where Ti: the mean tumor volume in the administration group, T0: the mean tumor volume in the administration group on D0 day, Vi: the mean tumor volume in the isotype control group, V0: the mean tumor volume in the isotype control group on D0 day . Determination of tumor volume: vernier calipers were used to measure the long diameter (a) and wide diameter (b) of the tumor, 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.

Dosage and mode of administration are shown in Table 15.

Table 15 Dosing trial design

编号Numbering	给药剂量Dosage	给药次数number of doses	给药方式way 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
Ab6Ab6	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection
Ab7Ab7	10mg/kg10mg/kg	Q3DX6Q3DX6	腹腔注射intraperitoneal injection

*Dosed every 3 days for a total of 6 times.

As shown in Figure 11A, the antitumor efficacy of binding proteins Ab0, Ab1, Ab6 and Ab7 in human Siglec15 transgenic mice was detected.

As shown in FIG. 11B , changes in body weight of human Siglec15 transgenic mice following administration of binding proteins Ab0, Ab1, Ab6 and Ab7.

As shown in Table 16, on the 24th day after inoculation, the antigen-binding proteins Ab0, Ab1, Ab6 and Ab7 of the present application significantly inhibited tumor growth at a dose of 10 mg/kg, and the tumor volume inhibition rates were 54%, 63%, 51% and 65%.

The above experiments showed that the antigen binding proteins Ab0, Ab1, Ab6 and Ab7 of the present application significantly inhibited tumor growth, and had no significant effect on the body weight of human Siglec15 transgenic mice.

Table 16 Antigen-binding proteins Ab0, Ab1, Ab6 and Ab7 of the present application on day 24 inhibit tumor growth in vivo

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

Example 12 Biological activity of humanized Siglec15 antigen-binding protein

The TRAP Staining kit (source: Cosmo Bio Co., LTD, product number: AK04F) was used to detect the activity of different Siglec15 antigen-binding proteins in inhibiting osteoclast formation.

(1) Quickly thaw the cryopreserved human peripheral mononuclear cells (PBMC) in a 37° water bath, and use the Pan Monocyte Isolation Kit (Source: Miltenyi, Item No.: 130-096-537) to sort the monocytes.

(2) Centrifugal counting, prepare 1640 complete medium with serum containing 25ng/ml M-SC (source: peprotech, product number: AF-300-25) and 30ng/ml RANKL (source: peprotech, product number: AF-310-01). , adjust the cell density and spread in 96-well plates.

(3) Add 100ul of antibody prepared in complete medium to each well, and the final concentration is 5ug/ml. No-RANKL medium and complete medium (containing M-CSF+RANKL) were set as control groups.

(4) Aspirate the original medium every two days, add fresh medium and antibodies, and continue culturing in the incubator.

(5) The supernatant was collected on the 8th day, incubated with 50ul of TRAP Staining Kit buffer and 10ul of supernatant for 2h, and the level of tartrate-resistant acid phosphatase secreted by osteoclasts was detected at a wavelength of 540nm.

The above experiments show that the Siglec15 antigen binding protein of the applicant can inhibit the formation of osteoclasts.

As shown in Figure 12, the humanized Siglec15 antigen-binding protein was able to inhibit the formation of osteoclasts, and the activity was comparable to that of the positive antibody.

As shown in Table 17, the humanized Siglec15 antigen-binding protein was able to inhibit the formation of osteoclasts.

Table 17 Humanized Siglec15 antigen binding protein inhibits the formation of osteoclasts

Example 13 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 the Siglec15 antigen-binding protein of the present invention in combination with PD-1 antibody.

Human PD-1/PD-L1 transgenic mice: Female C57BL/6 background human PD-1/PD-L1 transgenic mice (6 weeks) were purchased from Shanghai Biositu Jiangsu Gene Biotechnology Co., Ltd. Mice were acclimated for 7 days after arrival before the study began.

Cells: Mouse-derived MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. and routinely subcultured according to the instructions; MC38 cells were genetically modified to overexpress human-derived Siglec15, and the cell was named MC38/human Siglec15 cells for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in serum-free medium and adjusted for cell density. 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 cells. tumor mouse model.

Dosing:

On the 7th day of tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with tumor volume in the range of 120 mm ³ to 230 mm ³ were selected and grouped by tumor volume (6 mice in each group). Single dose, PD-1 antibody (Pembrolizumab, source: Taizhou Baiying Biotechnology Co., Ltd., product number: B2014-CHO); on the 7th, 10th, 14th, 17th, 21st, 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), antigen-binding protein of the application; during the administration period The changes of tumor volume and body weight of mice in each group were monitored, and the monitoring frequency was 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. The calculation formula is as follows: TGI(%)=[1-(Ti-T0)/(Vi-V0) ]×100; where Ti: the mean tumor volume in the administration group, T0: the mean tumor volume in the administration group on D0 day, Vi: the mean tumor volume in the isotype control group, V0: the mean tumor volume in the isotype control group on D0 day . Determination of tumor volume: vernier calipers were used to measure the long diameter (a) and wide diameter (b) of the tumor, 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.

Dosage and mode of administration are shown in Table 18.

Table 18 Dosing trial design

编号Numbering	给药剂量Dosage	给药次数number of doses	给药方式way of administration
阴性对照抗体Negative control antibody	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
PD-1抗体PD-1 antibody	1mg/kg1mg/kg	单次single	腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
Ab1Ab1	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
Ab7Ab7	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
PD-1抗体+阳性对照抗体PD-1 antibody + positive control antibody	1mg/kg+5mg/kg1mg/kg+5mg/kg	单次+BIWX6Single +BIWX6	腹腔注射intraperitoneal injection
PD-1抗体+Ab1PD-1 antibody + Ab1	1mg/kg+5mg/kg1mg/kg+5mg/kg	单次+BIWX6Single +BIWX6	腹腔注射intraperitoneal injection
PD-1抗体+Ab7PD-1 antibody + Ab7	1mg/kg+5mg/kg1mg/kg+5mg/kg	单次+BIWX6Single +BIWX6	腹腔注射intraperitoneal injection

*Dosed twice a week for a total of 6 times.

As shown in Figure 13A, the anti-tumor efficacy of antigen-binding proteins Ab1, Ab7 and PD-1 antibody in combination with human PD-1/PD-L1 transgenic mice was detected.

As shown in FIG. 13B , the body weight of human PD-1/PD-L1 transgenic mice was changed after the combination of antigen-binding proteins Ab1 and Ab7 with PD-1 antibody.

As shown in Table 19, on the 28th day after inoculation, the tumor volume inhibition rates of the antigen-binding proteins Ab1 and Ab7 of the present application were 5% and 14% at a dose of 5 mg/kg, respectively, and the PD-1 antibody at a dose of 1 mg/kg. The tumor volume inhibition rate was 39%; the tumor volume inhibition rate at the combined dose of Ab1 and PD-1 antibody was 50%; the tumor volume inhibition rate at the combined dose of Ab7 and PD-1 antibody was 61%; the antigen binding protein of this application Ab1 and Ab7, respectively, in combination with PD-1 antibody significantly inhibited tumor growth.

The above experiments show that the antigen binding proteins Ab1 and Ab7 of the present application in combination with 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 19 Antigen-binding protein Ab1, Ab7 and PD-1 antibody combination of the present application on day 28 inhibits tumor growth in vivo

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

Example 14 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 the Siglec15 antigen-binding protein of the present invention in combination with PD-L1 antibody.

Dosing:

On the 8th day of tumor cell inoculation, the tumor volume of each mouse was detected, and the mice with tumor volume in the range of 100 mm ³ to 200 mm ³ were selected and grouped by tumor volume (6 mice in each group). , 12, 15, 19, 22, 26 days of administration, negative control antibody (human IgG), PD-L1 antibody (atezolizumab, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number: HSP099-20) , positive control antibody (SPH-Sg-PC-1, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number HSP067-41), the antigen-binding protein of this application; monitor the tumor volume and body weight changes in each group of mice during administration, monitor The frequency is 2 times/week, and continuous monitoring is performed for 3 weeks. Body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 29th day after inoculation. The calculation formula is as follows: TGI(%)=[1-(Ti-T0)/(Vi-V0) ]×100; where Ti: the mean tumor volume in the administration group, T0: the mean tumor volume in the administration group on D0 day, Vi: the mean tumor volume in the isotype control group, V0: the mean tumor volume in the isotype control group on D0 day . Determination of tumor volume: vernier calipers were used to measure the long diameter (a) and wide diameter (b) of the tumor, 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.

Dosage and mode of administration are shown in Table 20.

Table 20 Dosing trial design

编号Numbering	给药剂量Dosage	给药次数number of doses	给药方式way of administration
阴性对照抗体Negative control antibody	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
PD-L1抗体PD-L1 antibody	3mg/kg3mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
阳性对照抗体positive control antibody	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
Ab1Ab1	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
Ab7Ab7	5mg/kg5mg/kg	BIWX6BIWX6	腹腔注射intraperitoneal injection
PD-L1抗体+阳性对照抗体PD-L1 antibody + positive control antibody	3mg/kg+5mg/kg3mg/kg+5mg/kg	BIWX6+BIWX6BIWX6+BIWX6	腹腔注射intraperitoneal injection
PD-L1抗体+Ab1PD-L1 antibody + Ab1	3mg/kg+5mg/kg3mg/kg+5mg/kg	BIWX6+BIWX6BIWX6+BIWX6	腹腔注射intraperitoneal injection
PD-L1抗体+Ab7PD-L1 antibody + Ab7	3mg/kg+5mg/kg3mg/kg+5mg/kg	BIWX6+BIWX6BIWX6+BIWX6	腹腔注射intraperitoneal injection

*Dosed twice a week for a total of 6 times.

As shown in Figure 14A, the antitumor efficacy of antigen-binding proteins Ab1 and Ab7 combined with PD-L1 antibody was detected in human PD-1/PD-L1 transgenic mice.

As shown in FIG. 14B , the body weight of human PD-1/PD-L1 transgenic mice was changed after the combination of antigen-binding proteins Ab1 and Ab7 with PD-L1 antibody.

As shown in Table 21, on the 29th day after inoculation, the tumor volume inhibition rates of the antigen-binding proteins Ab1 and Ab7 of the present application were 17% and -10%, respectively, at a dose of 5 mg/kg, and the inhibition rates of PD-L1 antibody at a dose of 3 mg/kg were respectively 17% and -10%. The tumor volume inhibition rate was 19% under the combined dose of Ab1 and PD-L1 antibody; the tumor volume inhibition rate was 64% under the combined dose of Ab7 and PD-L1 antibody; the tumor volume inhibition rate was 58% under the combined dose of Ab7 and PD-L1 antibody; The combination of protein Ab1 and Ab7 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 21 Antigen-binding protein Ab1, Ab7 and PD-L1 antibody combination of the present application on day 29 inhibits tumor growth in vivo

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

The foregoing detailed description has been presented by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments presently enumerated in this 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 the Biacore assay, it specifically binds to human Siglec15 protein with a KD value of about 4E-09M or less;

b) In flow assay, specifically binds to human Siglec15 expressed on the surface of CHOK1 cells with an EC50 value of about 0.1 μg/ml or less;

c) relieve the proliferation inhibition of PBMC in blood by Siglec15;

d) specifically binds to human Siglec15 in an ELISA assay with an EC50 value of about 0.2 μg/ml or less;

e) specifically binds to monkey Siglec15 in an ELISA assay with an EC50 value of about 0.1 μg/ml or less;

f) In an ELISA assay, specifically binds to murine Siglec15 with an EC50 value of about 0.1 μg/ml or less.
The isolated antigen-binding protein of claim 1, comprising HCDR3, said HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 13.
The isolated antigen binding protein of any one of claims 1-2, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO:9.
The isolated antigen-binding protein of any one of claims 1-3, comprising HCDR1 comprising the amino acid sequence shown in SEQ ID NO:4.
The isolated antigen binding protein of any one of claims 1-4, comprising a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3 comprising the HCDR3 set forth in SEQ ID NO:13 The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.
The isolated antigen-binding protein of any one of claims 1-5, comprising 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 -FR1 comprises the amino acid sequence shown in SEQ ID NO:64.
The isolated antigen-binding protein of claim 6, wherein the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.
The isolated antigen binding protein of any one of claims 1-7, 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:65.
The isolated antigen binding protein of claim 8, wherein the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5-8.
The isolated antigen binding protein of any one of claims 1-9, comprising H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 comprising SEQ ID The amino acid sequence shown in NO:66.
The isolated antigen-binding protein of claim 10, wherein the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.
The isolated antigen-binding protein of any one of claims 1-11, comprising H-FR4, the N-terminus of said H-FR4 is directly or indirectly linked to the C-terminus of said HCDR3, and the H-FR4 -FR4 comprises the amino acid sequence shown in SEQ ID NO:67.
The isolated antigen binding protein of claim 12, wherein the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 14 and 15.
The isolated antigen-binding protein of any one of claims 1-13, comprising H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NO: 64 The H-FR2 comprises the amino acid sequence shown in SEQ ID NO:65; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:66; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:67 amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-14, comprising H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising SEQ ID NOs: 1-3 The amino acid sequence shown in any one of the amino acid sequences; the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-8; the H-FR3 comprises any one of SEQ ID NOs: 10-12 and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 14 and 15.
The isolated antigen binding protein of any one of claims 14-15, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from any of the following groups:

a) H-FR1: SEQ ID NO: 1, H-FR2: SEQ ID NO: 5, H-FR3: SEQ ID NO: 10 and H-FR4: SEQ ID NO: 14;

b) H-FR1: SEQ ID NO: 2, H-FR2: SEQ ID NO: 6, H-FR3: SEQ ID NO: 11 and H-FR4: SEQ ID NO: 15;

c) H-FR1: SEQ ID NO: 2, H-FR2: SEQ ID NO: 7, H-FR3: SEQ ID NO: 12 and H-FR4: SEQ ID NO: 15;

d) H-FR1: SEQ ID NO:3, H-FR2: SEQ ID NO:8, H-FR3: SEQ ID NO:12 and H-FR4: SEQ ID NO:15.
The isolated antigen binding protein of any one of claims 1-16, comprising a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:72.
The isolated antigen binding protein of claim 17, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 29-32.
The isolated antigen-binding protein of any one of claims 1-18, comprising LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:26.
The isolated antigen binding protein of any one of claims 1-19, comprising LCDR2 comprising the amino acid sequence shown in SEQ ID NO:22.
The isolated antigen-binding protein of any one of claims 1-20, comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:19.
The isolated antigen-binding protein of any one of claims 1-21, comprising a light chain variable region VL, the VL comprising the LCDR1, LCDR2 and LCDR3, the LCDR3 comprising SEQ ID NO:26 The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 22; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19.
The isolated antigen-binding protein of any one of claims 1-22, comprising L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 -FR1 comprises the amino acid sequence shown in SEQ ID NO:68.
The isolated antigen-binding protein of claim 23, wherein the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 16-18.
The isolated antigen binding protein of any one of claims 1-24, comprising L-FR2, said L-FR2 being located between said LCDR1 and said LCDR2, and said L-FR2 comprising SEQ ID The amino acid sequence shown in NO:69.
The isolated antigen binding protein of claim 25, wherein the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20 and 21.
The isolated antigen binding protein of any one of claims 1-26, comprising L-FR3, said L-FR3 being located between said LCDR2 and said LCDR3, and said L-FR3 comprising SEQ ID The amino acid sequence shown in NO:70.
The isolated antigen-binding protein of claim 27, wherein the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23-25.
The isolated antigen-binding protein of any one of claims 1-28, comprising L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 -FR4 comprises the amino acid sequence shown in SEQ ID NO:71.
The isolated antigen-binding protein of claim 29, wherein the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27 and 28.
The isolated antigen binding protein of any one of claims 1-30, comprising L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising SEQ ID NO: 68 The L-FR2 comprises the amino acid sequence shown in SEQ ID NO:69; the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:70; and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:71 amino acid sequence shown.
The isolated antigen binding protein of any one of claims 1-31, comprising L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 comprising SEQ ID NOs: 16-18 The amino acid sequence shown in any one of the amino acid sequences; the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20 and 21; the L-FR3 comprises any one of SEQ ID NOs: 23-25 and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NOs: 27 and 28.
The isolated antigen-binding protein of any one of claims 31-32, wherein the L-FR1, L-FR2, L-FR3 and L-FR4 comprise an amino acid sequence selected from any of the following groups:

a) L-FR1: SEQ ID NO: 16, L-FR2: SEQ ID NO: 20, L-FR3: SEQ ID NO: 23 and L-FR4: SEQ ID NO: 27;

b) L-FR1: SEQ ID NO: 17, L-FR2: SEQ ID NO: 21, L-FR3: SEQ ID NO: 24 and L-FR4: SEQ ID NO: 28;

c) L-FR1: SEQ ID NO: 18, L-FR2: SEQ ID NO: 21, L-FR3: SEQ ID NO: 25 and L-FR4: SEQ ID NO: 28;

d) L-FR1: SEQ ID NO: 18, L-FR2: SEQ ID NO: 20, L-FR3: SEQ ID NO: 25 and L-FR4: SEQ ID NO: 28.
The isolated antigen binding protein of any one of claims 1-33, comprising a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:73.
The isolated antigen-binding protein of claim 34, wherein the VL comprises the amino acid sequence set forth in any one of SEQ ID NOs: 33-36.
The isolated antigen-binding protein of any one of claims 1-35, comprising VH and VL comprising amino acid sequences selected from any of the following groups:

a) VH: SEQ ID NO: 29 and VL: SEQ ID NO: 33;

b) VH: SEQ ID NO:30 and VL: SEQ ID NO:34;

c) VH: SEQ ID NO:30 and VL: SEQ ID NO:35;

d) VH: SEQ ID NO:30 and VL: SEQ ID NO:36;

e) VH: SEQ ID NO: 31 and VL: SEQ ID NO: 34;

f) VH: SEQ ID NO:31 and VL: SEQ ID NO:35;

g) VH: SEQ ID NO: 31 and VL: SEQ ID NO: 36;

h) VH: SEQ ID NO: 32 and VL: SEQ ID NO: 34;

i) VH: SEQ ID NO:32 and VL: SEQ ID NO:35;

j) VH: SEQ ID NO:32 and VL: SEQ ID NO:36.
The isolated antigen-binding protein of any one of claims 1-36, comprising a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.
The isolated antibody binding protein of claim 37, wherein the heavy chain constant region comprises a constant region derived from human IgG.
The isolated antigen binding protein of any one of claims 37-38, wherein the heavy chain constant region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39-42.
The isolated antigen-binding protein of any one of claims 1-39, comprising a light chain constant region, and the light chain constant region comprises an Igκ-derived constant region or an Igλ-derived constant region.
The isolated antigen binding protein of claim 40, wherein the light chain constant region comprises a constant region derived from human Igκ.
The isolated antigen binding protein of any one of claims 40-41, wherein the light chain constant region comprises the amino acid sequence set forth in any one of SEQ ID NO:43.
The isolated antigen-binding protein of any one of claims 1-42, which comprises an antibody or antigen-binding fragment thereof.
The isolated antigen-binding protein of claim 43, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv , VHH and/or dAb.
The isolated antigen binding protein of any one of claims 43-44, wherein the antibody is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
A polypeptide comprising the isolated antigen binding protein of any one of claims 1-45.
An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-45 or the polypeptide of claim 46.
An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-45, or the polypeptide of claim 46.
A vector comprising the isolated nucleic acid molecule of claim 48.
A cell comprising the isolated antigen binding protein of any one of claims 1-45, the polypeptide of claim 46, the immunoconjugate of claim 47, the isolated nucleic acid of claim 48 The molecule and/or the vector of claim 49.
A method for preparing the isolated antigen binding protein of any one of claims 1-45 or the polypeptide of claim 46, the method comprising remaking the isolated antigen of any one of claims 1-45 The cells of claim 50 are cultured under conditions in which the binding protein or the polypeptide of claim 46 is expressed.
A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-45, the polypeptide of claim 46, the immunoconjugate of claim 47, the isolated of claim 48 The nucleic acid molecule of claim 49, the cell of claim 50, and/or a pharmaceutically acceptable adjuvant and/or excipient.
A pharmaceutical combination comprising the isolated antigen binding protein of any one of claims 1-45 and an immune checkpoint inhibitor.
The pharmaceutical combination of claim 53, wherein the immune checkpoint inhibitor comprises a substance that inhibits PD-1/PD-L1 interaction.
The pharmaceutical combination of any one of claims 53-54, 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 of any one of claims 53-55, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody.
The pharmaceutical combination of claim 56, wherein the anti-PD-1 antibody comprises a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:46.
The pharmaceutical combination of any one of claims 56-57, wherein the anti-PD-1 antibody comprises a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:45.
The pharmaceutical combination of any one of claims 56-58, wherein the anti-PD-1 antibody comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:44.
The pharmaceutical combination of any one of claims 56-59, wherein the anti-PD-1 antibody comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, the HCDR3 comprising SEQ ID NO: The amino acid sequence shown in 46; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:45; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:44.
The pharmaceutical combination of any one of claims 56-60, wherein the anti-PD-1 antibody comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO:50.
The pharmaceutical combination of any one of claims 56-61, wherein the anti-PD-1 antibody comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:49.
The pharmaceutical combination of any one of claims 56-62, wherein the anti-PD-1 antibody comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:48.
The pharmaceutical combination of any one of claims 56-63, wherein the anti-PD-1 antibody comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:47.
The pharmaceutical combination of any one of claims 56-64, wherein the anti-PD-1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR3 comprises SEQ ID NO: 49; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:48; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:47.
The pharmaceutical combination of any one of claims 56-65, wherein the anti-PD-1 antibody comprises a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO:51.
The pharmaceutical combination of any one of claims 56-66, wherein the anti-PD-1 antibody comprises pembrolizumab.
The pharmaceutical combination of any one of claims 53-55, wherein the immune checkpoint inhibitor comprises an anti-PD-L1 antibody.
The pharmaceutical combination of claim 68, wherein the anti-PD-L1 antibody comprises a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:60.
The pharmaceutical combination of any one of claims 68-69, wherein the anti-PD-L1 antibody comprises a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:59.
The pharmaceutical combination of any one of claims 68-70, wherein the anti-PD-L1 antibody comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:58.
The pharmaceutical combination of any one of claims 68-71, wherein the anti-PD-L1 antibody comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 comprising SEQ ID NO: The amino acid sequence shown in 60; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:59; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:58.
The pharmaceutical combination of any one of claims 68-72, wherein the anti-PD-L1 antibody comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO:54.
The pharmaceutical combination of any one of claims 68-73, wherein the anti-PD-L1 antibody comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:63.
The pharmaceutical combination of any one of claims 68-74, wherein the anti-PD-L1 antibody comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:62.
The pharmaceutical combination of any one of claims 68-75, wherein the anti-PD-L1 antibody comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:61.
The pharmaceutical combination of any one of claims 68-76, wherein the anti-PD-L1 antibody comprises a light chain variable region VL, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR3 comprises SEQ ID NO: 63; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:62; and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:61.
The pharmaceutical combination according to any one of claims 68-77, wherein the anti-PD-L1 antibody comprises a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO:55.
The pharmaceutical combination of any one of claims 68-78, wherein the anti-PD-L1 antibody comprises atezolizumab.
The pharmaceutical combination of any one of claims 53-79, which may be a pharmaceutical composition.
A method for detecting or assaying Siglec15, the method comprising using the isolated antigen binding protein of any one of claims 1-45 or the polypeptide of claim 46.
A detection kit for Siglec15, comprising the isolated antigen-binding protein of any one of claims 1-45 or the polypeptide of claim 46.
Use of the isolated antigen-binding protein of any one of claims 1-45 or the polypeptide of claim 46 in the preparation of a kit.
A method of modulating an immune response comprising administering to a subject in need an effective amount of the isolated antigen binding protein of any one of claims 1-45, the polypeptide of claim 46, and claim 47 The immunoconjugate, the isolated nucleic acid molecule of claim 48, the carrier of claim 49, the cell of claim 50 and/or the pharmaceutical composition of any one of claims 52 , and/or a pharmaceutically acceptable therapeutic agent.
A method of modulating an immune response, comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination of any one of claims 53-80, and/or a pharmaceutically acceptable therapeutic agent.
The isolated antigen binding protein of any one of claims 1-45, the polypeptide of claim 46, the immunoconjugate of claim 47, the isolated nucleic acid molecule of claim 48, claim 49. The carrier of claim 50, the cell of claim 50 and/or the pharmaceutical composition of claim 52, for use in preventing, alleviating and/or treating a disease or disorder.
The pharmaceutical combination of any one of claims 53-80 for use in the prevention, alleviation and/or treatment of a disease or disorder.
The use of any one of claims 86-87, wherein the disease or disorder comprises abnormal bone metabolism.
The use of claim 88, wherein the abnormal bone metabolism comprises osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone metastases associated with multiple myeloma or cancer Destruction, osteopenia, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta.
The use of any one of claims 88-89, wherein the abnormal bone metabolism comprises osteoporosis.
The use of any one of claims 86-90, wherein the disease or disorder comprises a tumor.
The use of claim 91, wherein the tumor comprises a tumor associated with the expression of Siglec15.
The use of any one of claims 91-92, wherein the tumor comprises a solid tumor.
The use of any one of claims 91-93, wherein the tumor comprises colon cancer.
The isolated antigen binding protein of any one of claims 1-45, the polypeptide of claim 46, the immunoconjugate of claim 47, the isolated nucleic acid molecule of claim 48, claim Use of the carrier according to 49, the cell according to claim 50 and/or the pharmaceutical composition according to claim 52 in the preparation of a medicament for preventing and/or treating a disease or disorder.
Use of the pharmaceutical combination of any one of claims 53-80 in the manufacture of a medicament for preventing and/or treating a disease or condition.
The use of any one of claims 95-96, wherein the disease or disorder comprises abnormal bone metabolism.
The use of claim 97, wherein the abnormal bone metabolism comprises osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, bone metastases associated with multiple myeloma or cancer Destruction, osteopenia, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta.
The use of any one of claims 97-98, wherein the abnormal bone metabolism comprises osteoporosis.
The use of any one of claims 95-99, wherein the disease or disorder comprises a tumor.
The use of claim 100, wherein the tumor comprises a tumor associated with the expression of Siglec15.
The use of any one of claims 100-101, wherein the tumor comprises a solid tumor.
The use of any one of claims 100-102, wherein the tumor comprises colon cancer.
A method of preventing and/or treating a disease or condition, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein of any one of claims 1-45, the antigen-binding protein of claim 46 A polypeptide, the immunoconjugate of claim 47, the isolated nucleic acid molecule of claim 48, the vector of claim 49, and/or the cell of claim 50.
A method of preventing and/or treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of the pharmaceutical combination of any one of claims 53-80.
The method of any one of claims 104-105, wherein the disease or disorder comprises abnormal bone metabolism.
The method of claim 106, wherein the abnormal bone metabolism comprises osteoporosis, bone destruction with rheumatoid arthritis, cancerous hypercalcemia, bone metastases with multiple myeloma or cancer Destruction, osteopenia, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis imperfecta.
The method of claims 106-107, wherein the abnormal bone metabolism comprises osteoporosis.
The method of any one of claims 104-108, wherein the disease or disorder comprises a tumor.
The method of claim 109, wherein the tumor comprises a tumor associated with the expression of Siglec15.
The method of any one of claims 109-110, wherein the tumor comprises a solid tumor.
The method of any one of claims 109-111, wherein the tumor comprises colon cancer.