Preparation of Siglec-15 binding proteins and uses thereof

Abstract

The present invention provides preparations of Siglec-15 binding proteins, and in particular relates to isolated antigen binding proteins comprising HCDR3. Use of the antigen binding proteins in the prevention and treatment of diseases is provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present application relates to the biomedical field, and in particular to the preparation of Siglec-15 binding proteins and their uses. [Background technology]

[0002] 2. Background of the Invention Siglecs (sialic acid-binding immunoglobulin-like lectins), as a kind of classical immunoglobulin-like lectins, can regulate immune balance in vivo. There are as many as 30 members in the Siglec family, which are expressed on the cell surface. Abnormalities of several molecules of this family member are associated with many diseases, and Siglec-15 is one of them. Siglec-15 does not contain an immunoreceptor tyrosine-based inhibitiory motif (ITIM) or ITIM-like sequence in the intracellular region, and intracellular Siglec-15 is associated with DAP12 or DAP10, which contain an immunoreceptor tyrosine-based activating motif (ITAM) signal, as shown by experimental data. However, most of the intracellular segments of Siglecs contain an immunoreceptor tyrosine-based inhibitiory motif, thereby exerting immunosuppressive functions. It has been shown that Siglec-15 can bind to sialyl-Tn and Neu5 Aca2-6GalNAca, suggesting that this may be a ligand for Siglec-15. Siglec-15 is mainly expressed in myeloid cells in normal humans. Siglec-15 is abnormally highly expressed in various solid tumors. Preclinical in vitro studies and animal tumor models have shown that Siglec-15 antibodies can activate T cells and suppress tumors. However, there is currently a lack of suitable Siglec-15 antibodies for tumor therapy, and there is an urgent need in the art to develop more Siglec-15-binding proteins with therapeutic effects. Summary of the Invention

[0003] Summary of the Invention The present application provides a Siglec-15 binding protein. The Siglec-15 binding protein of the present application has (1) a K D(2) having the ability to bind to Siglec-15 or a functional fragment thereof with an EC value of about 3.25 nM or less. 50 (3) having good physicochemical properties such as thermal stability; (4) having an effect on immune cell proliferation, e.g., having an effect of reversing the inhibition of immune cell proliferation caused by Siglec-15; (5) having tumor-suppressing ability, e.g., being able to suppress tumors with a tumor volume inhibition rate of about 23% or more; and (6) having an effect on cytokine secretion.

[0004] In one aspect, the present application provides an isolated antigen binding protein comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1.

[0005] In some embodiments, the isolated antigen binding protein comprises an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:33.

[0006] In some embodiments, the isolated antigen binding protein comprises an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0007] In some embodiments, the isolated antigen binding protein comprises an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:34.

[0008] In some embodiments, the isolated antigen binding protein comprises an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

[0009] In some embodiments, the isolated antigen binding protein comprises an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9.

[0010] In some embodiments, the isolated antigen binding protein comprises an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:35.

[0011] In some embodiments, the isolated antigen binding protein comprises an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0012] In some embodiments, the isolated antigen binding protein comprises an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:36.

[0013] In some embodiments, the isolated antigen binding protein comprises an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID NO:14.

[0014] In some 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 H-FR1 comprises the amino acid sequence as set forth in SEQ ID NO:15.

[0015] In some embodiments, the isolated antigen binding protein comprises H-FR2, wherein H-FR2 is located between HCDR1 and HCDR2, and wherein H-FR2 comprises the amino acid sequence as set forth in SEQ ID NO:16.

[0016] In some embodiments, the isolated antigen binding protein comprises H-FR3, wherein H-FR3 is located between HCDR2 and HCDR3, and wherein H-FR3 comprises the amino acid sequence as set forth in SEQ ID NO:17.

[0017] In some embodiments, the isolated antigen binding protein comprises H-FR4, wherein the N-terminus of H-FR4 is linked directly or indirectly to the C-terminus of HCDR3, and wherein H-FR4 comprises the amino acid sequence as set forth in SEQ ID NO:18.

[0018] In some embodiments, the isolated antigen binding protein comprises L-FR1, the C-terminus of L-FR1 being directly or indirectly linked to the N-terminus of LCDR1, and L-FR1 comprising an amino acid sequence as set forth in SEQ ID NO:19.

[0019] In some embodiments, the isolated antigen binding protein comprises L-FR2, wherein L-FR2 is located between LCDR1 and LCDR2, and wherein L-FR2 comprises the amino acid sequence as set forth in SEQ ID NO:20.

[0020] In some embodiments, the isolated antigen binding protein comprises L-FR3, wherein L-FR3 is located between LCDR2 and LCDR3, and wherein L-FR3 comprises the amino acid sequence as set forth in SEQ ID NO:21.

[0021] In some embodiments, the isolated antigen binding protein comprises an L-FR4, wherein the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises an amino acid sequence as set forth in SEQ ID NO:22.

[0022] In some embodiments of the isolated antigen binding protein, HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:33, and HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:34.

[0023] In some embodiments of the isolated antigen binding protein, HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, and HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.

[0024] In some embodiments of the isolated antigen binding protein, LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:35, and LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:36.

[0025] In some embodiments of the isolated antigen binding protein, LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID NO:14.

[0026] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:33, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:34, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:35, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:36.

[0027] In some embodiments of the isolated antigen binding protein, HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, HCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, LCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and LCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID NO:14.

[0028] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:2, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0029] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:2, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:8, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0030] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:3, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0031] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:4, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0032] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:5, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0033] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:6, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0034] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:2, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:10, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:14.

[0035] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:3, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:11, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0036] In some embodiments of the isolated antigen binding protein, HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:3, HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:7, LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:9, LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO:12, and LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO:13.

[0037] In some embodiments, the isolated antigen binding protein comprises a heavy chain variable region (VH) comprising the amino acid sequence as set forth in SEQ ID NO:37.

[0038] In some embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence as set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

[0039] In some embodiments, the isolated antigen binding protein comprises a light chain variable region (VL) comprising the amino acid sequence as set forth in SEQ ID NO:38.

[0040] In some embodiments, the isolated antigen binding protein comprises a VL comprising the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.

[0041] In some embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence as set forth in SEQ ID NO:37 and a VL comprising the amino acid sequence as set forth in SEQ ID NO:38.

[0042] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28, and a VL comprising an amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.

[0043] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:23, and a VL comprising an amino acid sequence as set forth in SEQ ID NO:29.

[0044] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:24 and a VL comprising an amino acid sequence as set forth in SEQ ID NO:29.

[0045] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:25 and a VL comprising an amino acid sequence as set forth in SEQ ID NO:29.

[0046] In some embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence as set forth in SEQ ID NO:26 and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29.

[0047] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:27 and a VL comprising an amino acid sequence as set forth in SEQ ID NO:29.

[0048] In some embodiments, the isolated antigen binding protein comprises a VH comprising the amino acid sequence as set forth in SEQ ID NO:28 and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29.

[0049] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:23 and a VL comprising an amino acid sequence as set forth in SEQ ID NO:30.

[0050] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:25 and a VL comprising an amino acid sequence as set forth in SEQ ID NO:31.

[0051] In some embodiments, the isolated antigen binding protein comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO:25 and a VL comprising an amino acid sequence as set forth in SEQ ID NO:32.

[0052] In some embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

[0053] In some embodiments of the isolated antigen binding protein, the antibody heavy chain constant region comprises a heavy chain constant region derived from a human antibody.

[0054] In some embodiments of the isolated antigen binding protein, the antibody heavy chain constant region comprises a heavy chain constant region derived from an IgG.

[0055] In some embodiments of the isolated antigen binding protein, the antibody heavy chain constant region comprises a heavy chain constant region derived from IgG1.

[0056] In some embodiments, the isolated antigen binding protein comprises an antibody light chain constant region.

[0057] In some embodiments of the isolated antigen binding protein, the antibody light chain constant region comprises a light chain constant region derived from a human antibody.

[0058] In some embodiments of the isolated antigen binding protein, the antibody light chain constant region comprises a constant region derived from human Igκ.

[0059] In some embodiments, the isolated antigen binding protein comprises an antibody or an antigen-binding fragment thereof.

[0060] In some embodiments of the isolated antigen binding protein, the antibody comprises a monoclonal antibody.

[0061] In some embodiments of the isolated antigen binding protein, the antibody is selected from one or more of the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0062] In some embodiments of the isolated antigen binding protein, the antigen binding fragment is selected from one or more of the group consisting of a Fab, a Fab', an Fv fragment, F(ab')2, F(ab)2, a scFv, a di-scFv, a VHH, and a dAb.

[0063] In some embodiments, the isolated antigen binding protein is capable of specifically binding to a sialic acid-binding immunoglobulin-like lectin (Siglec) or a functionally active fragment thereof.

[0064] In some embodiments of the isolated antigen binding protein, the Siglec comprises Siglec-15.

[0065] In some embodiments of the isolated antigen binding protein, the Siglec comprises a human Siglec, a monkey Siglec, and / or a mouse Siglec.

[0066] In some embodiments, the isolated antigen binding protein has a K of about 1.56E-10 M or less.D The antibody has the ability to bind to Siglec-15 or a functional fragment thereof at a specific antibody level.

[0067] In some embodiments, the isolated antigen binding protein has an EC 50 The antibody has the ability to bind to Siglec-15 or a functional fragment thereof at a specific antibody level.

[0068] In some embodiments of the isolated antigen binding protein, the Siglec-15 or functional fragment thereof comprises Siglec-15 or a functional fragment thereof expressed on a cell.

[0069] In some embodiments, the isolated antigen binding protein has the ability to affect the proliferation of immune cells.

[0070] In some embodiments of the isolated antigen binding protein, affecting immune cell proliferation comprises reducing Siglec-15 associated inhibition of immune cell proliferation.

[0071] In some embodiments of the isolated antigen binding protein, the immune cell comprises an immune cell from a human.

[0072] In some embodiments of the isolated antigen binding protein, the immune cell comprises a lymphocyte.

[0073] In some embodiments of the isolated antigen binding protein, the immune cells comprise peripheral blood lymphocytes.

[0074] In some embodiments of the isolated antigen binding protein, the immune cell comprises a T cell.

[0075] In some embodiments of the isolated antigen binding protein, the immune cell is CD4 + Cells and / or CD8 + Contains cells.

[0076] In some embodiments, the isolated antigen binding protein has tumor suppressor capability.

[0077] In some embodiments of the isolated antigen binding protein, the tumor suppressive ability comprises affecting tumor volume.

[0078] In some embodiments, the isolated antigen binding protein has the ability to inhibit tumors with a tumor volume inhibition rate of about 23% or greater.

[0079] In some embodiments, the isolated antigen binding protein has the ability to affect cytokine secretion.

[0080] In some embodiments of the isolated antigen binding protein, secretion includes in vivo, ex vivo and / or in vitro secretion.

[0081] In some embodiments of the isolated antigen binding protein, cytokine secretion comprises lipopolysaccharide-induced cytokine secretion.

[0082] In some embodiments of the isolated antigen binding protein, the cytokine comprises an inflammatory factor.

[0083] In some embodiments of the isolated antigen binding protein, the cytokine comprises TNF-α and / or IL-6.

[0084] In another aspect, the present application also provides a polypeptide comprising an isolated antigen binding protein of the present application.

[0085] In some embodiments, the polypeptide comprises a fusion protein.

[0086] In another aspect, the present application also provides nucleic acid molecules encoding the isolated antigen binding proteins of the present application and / or the polypeptides of the present application.

[0087] In another aspect, the present application also provides a vector comprising the nucleic acid molecule of the present application.

[0088] In another aspect, the present application also provides an immunoconjugate comprising the isolated antigen-binding protein of the present application and / or the polypeptide of the present application.

[0089] In another aspect, the present application also provides a cell comprising and / or a cell that comprises an isolated antigen binding protein of the present application, a cell that comprises and / or expresses a polypeptide of the present application, a cell that comprises a nucleic acid molecule of the present application, a cell that comprises a vector of the present application, and / or a cell that comprises an immunoconjugate of the present application.

[0090] In another aspect, the present application also provides a pharmaceutical composition comprising an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, and / or a cell of the present application, and optionally a pharma- ceutically acceptable vehicle.

[0091] In another aspect, the present application also provides pharmaceutical combinations comprising an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, and / or a pharmaceutical composition of the present application.

[0092] In another aspect, the present application also provides kits comprising an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, and / or a pharmaceutical combination of the present application.

[0093] In some embodiments, the kit is used to detect the presence and / or amount of Siglecs in a sample.

[0094] In some embodiments of the kit, the Siglec comprises Siglec-15.

[0095] In another aspect, the present application also provides a method of preparing an antigen binding protein and / or polypeptide of the present application comprising culturing under conditions that allow expression of the antigen binding protein and / or polypeptide.

[0096] In some embodiments, the method comprises culturing the cells under conditions that allow expression of the antigen binding protein.

[0097] In some embodiments, the method comprises culturing the cells under conditions that allow expression of the polypeptide.

[0098] In another aspect, the present application also provides a method of detecting Siglec in a sample, comprising administering an isolated antigen binding protein of the present application, administering a polypeptide of the present application, administering a nucleic acid molecule of the present application, administering a vector of the present application, administering an immunoconjugate of the present application, administering a cell of the present application, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or using a kit of the present application.

[0099] In some embodiments of this method, the Siglec comprises Siglec-15.

[0100] In another aspect, the present application also provides a method of affecting binding of a Siglec, or a functionally active fragment thereof, to its ligand, comprising administering an isolated antigen binding protein of the present application, administering a polypeptide of the present application, administering a nucleic acid molecule of the present application, administering a vector of the present application, administering an immunoconjugate of the present application, administering a cell of the present application, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or using a kit of the present application.

[0101] In some embodiments, the methods include in vivo, ex vivo and / or in vitro methods.

[0102] In some embodiments of this method, the Siglec, or a functionally active fragment thereof, comprises Siglec-15, or a functionally active fragment thereof.

[0103] In some embodiments of this method, the ligand for the Siglec comprises leucine-rich repeat-containing protein 4C and / or sialylated Tn (sialyl-Tn).

[0104] In another aspect, the present application also provides a method of affecting immune cell proliferation comprising administering an isolated antigen binding protein of the present application, administering a polypeptide of the present application, administering a nucleic acid molecule of the present application, administering a vector of the present application, administering an immunoconjugate of the present application, administering a cell of the present application, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or using a kit of the present application.

[0105] In some embodiments, the methods include in vivo, ex vivo and / or in vitro methods.

[0106] In some embodiments of the method, affecting immune cell proliferation comprises reducing Siglec-15 associated inhibition of immune cell proliferation.

[0107] In some embodiments of this method, the immune cells comprise human-derived immune cells.

[0108] In some embodiments of the method, the immune cells comprise lymphocytes.

[0109] In some embodiments of this method, the immune cells comprise peripheral blood lymphocytes.

[0110] In some embodiments of this method, the immune cells comprise T cells.

[0111] In some embodiments of this method, the immune cells are CD4 + Cells and / or CD8 + Contains cells.

[0112] In another aspect, the present application also provides a method of affecting cytokine secretion comprising administering an isolated antigen binding protein of the present application, administering a polypeptide of the present application, administering a nucleic acid molecule of the present application, administering a vector of the present application, administering an immunoconjugate of the present application, administering a cell of the present application, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or using a kit of the present application.

[0113] In some embodiments of this method, the secretion comprises in vivo, ex vivo and / or in vitro secretion.

[0114] In some embodiments of this method, the cytokine secretion comprises lipopolysaccharide-associated cytokine secretion.

[0115] In some embodiments of this method, the cytokine comprises an inflammatory factor.

[0116] In some embodiments of this method, the cytokine comprises TNF-α and / or IL-6.

[0117] In another aspect, the present application also provides the use of an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, and / or a pharmaceutical combination of the present application in the manufacture of a kit.

[0118] In some embodiments of this use, the kit is used to detect the presence and / or amount of Siglecs in a sample.

[0119] In some embodiments of this use, the Siglec comprises Siglec-15.

[0120] In another aspect, the present application also provides the use of an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, a pharmaceutical combination of the present application, and / or a kit of the present application in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition.

[0121] In some embodiments of this use, the disease and / or condition comprises a tumor.

[0122] In some embodiments of this use, the tumor comprises a Siglec-associated tumor.

[0123] In some embodiments of this use, the Siglec comprises Siglec-15.

[0124] In some embodiments of this use, the disease and / or condition comprises a solid tumor.

[0125] In some embodiments of this use, the disease and / or condition comprises colon cancer.

[0126] In another aspect, the present application also provides an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, a pharmaceutical combination of the present application, and / or a kit of the present application for use in the prevention and / or treatment of a disease and / or condition.

[0127] In some embodiments, the disease and / or condition comprises a tumor.

[0128] In some embodiments, the tumor comprises a Siglec-associated tumor.

[0129] In some embodiments, the Siglec comprises Siglec-15.

[0130] In some embodiments, the disease and / or disorder comprises a solid tumor.

[0131] In some embodiments, the disease and / or disorder comprises colon cancer.

[0132] In another aspect, the present application also provides a method for the prevention and / or treatment of a disease and / or condition, comprising administering to a subject in need thereof an isolated antigen binding protein of the present application, a polypeptide of the present application, a nucleic acid molecule of the present application, a vector of the present application, an immunoconjugate of the present application, a cell of the present application, a pharmaceutical composition of the present application, a pharmaceutical combination of the present application, and / or a kit of the present application.

[0133] In some embodiments, the disease and / or condition comprises a tumor.

[0134] In some embodiments, the tumor comprises a Siglec-associated tumor.

[0135] In some embodiments, the Siglec comprises Siglec-15.

[0136] In some embodiments, the disease and / or disorder comprises a solid tumor.

[0137] In some embodiments, the disease and / or disorder comprises colon cancer.

[0138] Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. The following detailed description shows and describes merely exemplary embodiments of the present application. As will be recognized by those skilled in the art, the contents of the present application will enable those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the present application to which it pertains. Accordingly, the drawings and descriptions herein are to be considered as illustrative in nature and not restrictive.

[0139] Particular features of the present application to which the present application pertains are set forth in the appended claims. The features and advantages of the present application to which the present application pertains will be better understood with reference to the exemplary embodiments and drawings described in detail below, the brief description of which follows. [Brief description of the drawings]

[0140] [Figure 1] FIG. 1 shows a schematic diagram of the present yeast display library construction method. [Diagram 2] FIG. 2 shows the results of detection of the binding of the Siglec-15-binding protein of the present application to human Siglec-15 on the cell surface. [Diagram 3] FIG. 3 shows the results of detection of the binding of the Siglec-15-binding protein of the present application to monkey Siglec-15 on the cell surface. [Figure 4] FIG. 4 shows the results of detection of the binding of the Siglec-15-binding protein of the present application to mouse Siglec-15 on the cell surface. [Diagram 5] FIG. 5 shows the results of detection of the reversal of the growth inhibitory effect of Siglec-15 protein on CD4+ T cells by the Siglec-15-binding protein of the present application. [Figure 6] FIG. 6 shows the results of detection of the reversal of the growth inhibitory effect of Siglec-15 protein on CD8+ T cells by the Siglec-15-binding protein of the present application. [Figure 7] FIG. 7 shows the results of detection of the inhibitory effect of the Siglec-15 binding protein of the present application on tumor growth. [Figure 8] FIG. 8 shows the detection results regarding the effect of the Siglec-15 binding protein of the present application on the secretion level of TNF-α (TNF-alpha) 2 hours after LPS injection. [Figure 9] FIG. 9 shows the detection results regarding the effect of the Siglec-15 binding protein of the present application on the secretion level of TNF-α (TNF-alpha) 6 hours after LPS injection. [Figure 10]FIG. 10 shows the detection results regarding the effect of the Siglec-15 binding protein of the present invention on the secretion level of IL-6 2 hours after LPS injection. [Figure 11] FIG. 11 shows the detection results regarding the effect of the Siglec-15 binding protein of the present invention on the secretion level of IL-6 6 hours after LPS injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] Detailed Description of Specific Embodiments Other advantages and benefits of the present application will become readily apparent to those skilled in the art from the following description of specific embodiments.

[0142] Definition of Terms In this application, the term "sialic acid-binding immunoglobulin-like lectin", "sialic acid-binding Ig-like lectin" or "Siglec" generally refers to an immunoglobulin-like lectin that may have the ability to recognize sugar structures containing sialic acid. In vitro, Siglec-15 recombinant protein can suppress the proliferation and secretion of IFN-γ in human and mouse T cells, and can also suppress ovalbumin (OVA) antigen-activated OT-1 T cells. Siglec-15 is lowly expressed in macrophages and shows significant upregulation of expression after stimulation with macrophage colony-stimulating factor M-CSF. Compared with normal macrophages, Siglec-15 gene knockout macrophages can further induce T cell proliferation, indicating that Siglec-15 can directly suppress T cell activity. Preclinical in vitro studies and animal tumor models have shown that Siglec-15 antibodies or Siglec-15 binding proteins can activate T cells and suppress tumors. For example, the Siglec can be Siglec-15, which can have Uniprot Accession No. Q6ZMC9. The Siglec proteins of the present application can also encompass functionally active fragments thereof, and are not limited to proteins comprising functionally active fragments of Siglec proteins that result from processing and / or modifications that occur within the cell.

[0143] In the present application, the term "leucine-rich repeat-containing protein 4C" or "LRRC 4C" is used interchangeably and generally refers to a protein rich in repeat leucine sequences. LRRC4 may act as a ligand for Siglect-15, binding to and activating Siglec-15. LRRC4C may have the Uniprot accession number Q9HCJ2. The LRRC4C protein of the present application may also include functionally active fragments thereof, and is not limited to proteins comprising functionally active fragments of LRRC4C protein resulting from processing and / or modification occurring in cells.

[0144] In the present application, the term "sialylated Tn" or "sialyl-Tn" generally refers to a Tn antigen containing a sialyl group. The antigen may be an N-acetylgalactosamine (GalNAc) oligosaccharide linked to serine or threonine via a glycosidic bond. Sialyl-Tn expression may result from the activation of an aberrant glycosylation pathway, which generally occurs in tumor cells. The sialylated Tn protein of the present application may also include its functionally active fragments.

[0145] In this application, the term "lipopolysaccharide" or "LPS" generally refers to a substance composed of lipids and polysaccharides. Lipopolysaccharides are generally components of the outer cell wall of gram-negative bacteria. Lipopolysaccharides can stimulate the synthesis and / or secretion of various cytokines by human cells to induce an immune response while acting on the human body.

[0146] In this application, the term "functionally active fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment may retain or partially retain the ability of the full-length protein to bind to another molecule. For example, a functionally active fragment of a Siglec may retain or partially retain the function of the full-length Siglec to recognize sialic acid-containing sugar structures and / or suppress the function of the immune system.

[0147] As used herein, the term "secretion" generally refers to the transfer of an expressed polypeptide or protein by a cell to the extracellular environment. For example, as used herein, it may refer to the transfer of an expressed cytokine by an immune cell to the extracellular environment.

[0148] In this application, the term "cytokine" generally refers to a protein released by a cell population that acts on another cell as an intercellular modulator. The cytokine of this application can be an interleukin (IL), such as IL-6, tumor necrosis factor, such as TNF-α, and other polypeptide factors. In this application, the cytokine of this application can include functionally active fragments thereof, and can also include proteins from natural sources or recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

[0149] In the present application, the term "inflammatory factor" generally refers to a cytokine that promotes inflammation. In the present application, the inflammatory factor may include interleukin IL-6, and / or tumor necrosis factor TNF-α. In the present application, the inflammatory cytokine may include a functionally active fragment thereof, and may also include a protein from a natural source or from recombinant cell culture, as well as a biologically active equivalent of the native sequence cytokine.

[0150] In the present application, the term "IL-6" generally refers to interleukin. The IL-6 of the present application can promote inflammation. The IL-6 can be that having Uniprot accession number P05231. The IL-6 protein of the present application can also include functionally active fragments thereof, and is not limited to proteins comprising functionally active fragments of the IL-6 protein resulting from processing and / or modification occurring within the cell.

[0151] In this application, the term "TNFα" or "TNF-α" generally refers to tumor necrosis factor alpha. TNF-α can be a major mediator of inflammatory, immune and pathophysiological responses. TNF-α can include wild-type TNF-α, polymorphic variants of TNF-α, and functional equivalents of TNF-α from various species (e.g., human, mouse, and monkey). TNF-α can be that which has been granted Uniprot accession number P01375. The TNF-α protein of this application can also encompass functionally active fragments thereof, and is not limited to proteins comprising functionally active fragments of TNF-α protein resulting from processing and / or modification occurring within the cell.

[0152] As used herein, the term "lymphocyte" generally refers to white blood cells found in blood, lymph, and lymphoid tissues. For example, lymphocytes can include any monocyte and / or non-phagocytic white blood cell.

[0153] As used herein, the term "immune cells" generally refers to cells involved in an immune response. Immune cells may include T cells, B cells, natural killer (NK) cells, mast cells, and / or bone marrow derived phagocytes.

[0154] As used herein, the term "peripheral blood lymphocytes" generally refers to mature lymphocytes that circulate in the blood and are not localized to organs such as the spleen or lymph nodes. Peripheral blood lymphocytes can include T cells, NK cells, and / or B cells.

[0155] In this application, the term "T cells" generally refers to cells derived from the thymus. T cells that can participate in various cell-mediated immune responses include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cell populations include, but are not limited to, helper T cells (Th; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 -T cells or any other subpopulation of T cells are included.

[0156] In this application, the term "CD4" generally refers to the cluster of differentiation 4 protein expressed by cells. CD4 can be a co-receptor for T cell recognition of antigens and can bind to non-polypeptide regions of MHC class II molecules. T cells expressing CD4 (CD4 + T cells or CD4 positive T cells) can differentiate into helper T cells (Th) upon activation and can regulate immune responses. CD4 can be that which has been given the Uniprot accession number P01730. The CD4 protein of the present application can also encompass functionally active fragments thereof and is not limited to proteins comprising functionally active fragments of the CD4 protein resulting from processing and / or modification that occurs within the cell.

[0157] In this application, the term "CD8" generally refers to the Cluster of Differentiation 8 protein expressed by cells. CD8 can be a co-receptor for T cell recognition antigens and can bind to non-polypeptide regions of MHC class II molecules. T cells expressing CD8 (CD8 + T cells or CD8 positive T cells) can differentiate into cytotoxic T cells (CTLs) upon activation and can specifically kill target cells. CD8 can be that having Uniprot accession number P10966. The CD8 protein of the present application can also include functionally active fragments thereof and is not limited to proteins comprising functionally active fragments of the CD8 protein resulting from processing and / or modification occurring within the cell.

[0158] As used herein, the term "in vivo" generally refers to events that take place in a subject in vivo.

[0159] As used herein, the term "in vitro" generally refers to events that take place in a subject in vitro.

[0160] In this application, the term "ex vivo" generally refers to events involving treatment or surgery of cells, tissues and / or organs that have been removed from the subject's body. In one embodiment, these cells, tissues and / or organs can be returned to the subject's body via surgery or treatment.

[0161] In this application, the term "EC 50 "Effective concentration" or "EC50 value" generally refers to the 50% between the baseline value and the maximum response of a binding agent (e.g., an antibody) for an in vitro or in vivo assay, and is the 50% effective concentration, EC 50 Lower values ​​may indicate higher drug affinity and efficacy.

[0162] In this application, the term "K D "KD value" or "KD value" generally refers to a dissociation constant that can be determined by surface plasmon resonance (SPR). Generally, in an SPR analysis, real-time binding interactions between a ligand (a substance immobilized on a biosensor matrix) and an analyte (a substance in solution) are measured by SPR using a BIAcore system (Pharmacia Biosenser, Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (a substance on a biosensor matrix) and providing a ligand.

[0163] In the present application, the term "tumor suppressive ability" generally refers to any measurable inhibitory ability with respect to tumor cell proliferation in vitro or tumor growth in vivo, such as a reduction of at least 5% (which may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, e.g., at least 100%) of tumor cell proliferation in vitro or tumor growth in vivo.

[0164] In this application, the term "isolated" generally refers to a material obtained from a natural state by artificial means. For example, a non-isolated polynucleotide or polypeptide naturally occurs in a living animal, and the same polynucleotide or polypeptide isolated in high purity from its natural state can be said to be isolated. The term "isolated" does not exclude the inclusion of artificial or synthetic substances, nor the presence of other impurities that do not affect the activity of the material.

[0165] In the present application, the term "isolated antigen-binding protein" generally refers to a protein having antigen-binding ability, removed from its natural state. The "isolated antigen-binding protein" of the present application may comprise an antigen-binding moiety, and optionally a framework or framework portion that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding moiety to the antigen. The antigen-binding protein may comprise, for example, a protein framework region (FR) derived from an antibody or another protein framework region or an artificial framework region onto which CDRs or CDR derivatives have been grafted. Such frameworks may include, but are not limited to, an antibody-derived framework region comprising, for example, a mutation introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as a completely synthetic framework region comprising, for example, a biocompatible polymer. Examples of antigen binding proteins may include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, VHH, IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; and / or IgG4 antibodies and fragments thereof.

[0166] In this application, the term "CDR" is also referred to as "complementarity determining region" and generally refers to a region in an antibody variable domain that is highly variable in sequence and / or can form a structurally defined loop. For example, an antibody may comprise six CDRs, three in VH (HCDR1, HCDR2, and HCDR3) and three in VL (LCDR1, LCDR2, and LCDR3). In some embodiments, a natural camelid antibody consisting of only heavy chains can function normally and stably even without the presence of light chains. The CDRs of an antibody can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia comprehensive consideration, etc. Such coding systems are known in the art. See, for example, www.bioinf.org.uk / abs / index.html#kabatnum. For example, numbering of the amino acid sequence of an antigen-binding protein may be according to the IMGT numbering scheme (IMGT, IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al.,2000 Nucleic Acids Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310; Lefranc et al., 2005, DevComp Immunol 29: 185-203).For example, the CDRs of an antigen-binding protein can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY AcadSci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No.91-3242).

[0167] In this application, the term "FR" generally refers to the more conserved parts of antibody variable domains, called framework regions. For example, the variable domains of natural heavy and light chains may each comprise four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0168] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and / or light chain. The variable domains of the heavy and light chains are respectively referred to as "VH" and "VL" (or "VH", respectively). H " and "V LThese domains may generally be the most diverse parts of an antibody (compared to other antibodies of the same type) and may comprise the antigen-binding site. In this application, the term "variable" generally means that certain segments of the variable domains may differ substantially in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, variability may not be uniformly distributed in the variable domains. The variability is generally concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more conserved parts of the variable domains may be called framework regions (FRs). Naturally occurring heavy and light chain variable domains each comprise four FR regions, which are largely in a β-sheet configuration and connected by three CDRs to form loop junctions and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions, and together with the CDRs of the other chain, may facilitate the formation of the antigen-binding site of the antibody.

[0169] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide that contains an antigen-binding site, whether produced in vitro or in vivo. The term may include, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, nonspecific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, and grafted antibodies. For purposes of the present invention, unless modified by the term "intact", such as "intact antibody", the term "antibody" may also include antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that retain antigen-binding function (e.g., bind specifically to human Siglec). Generally, such fragments may comprise an antigen-binding domain. The basic four-chain antibody unit may be a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies may consist of five basic heterotetrameric units together with another polypeptide called the J chain and contain ten antigen-binding sites, whereas IgA antibodies may comprise two to five basic four-chain units that can be polymerized together with the J chain to form multivalent combinations. For IgG, the four-chain unit may generally be about 150,000 daltons. Each L chain may be linked to the H chain through one covalent disulfide bond, and the two H chains may be linked to each other by one or more disulfide bonds depending on the H chain isotype. Each of the H and L chains may also have regularly spaced intrachain disulfide bridges. Each H chain may have a heavy chain variable region (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha (α) and gamma (γ) chains, and four CH domains for the mu (μ) and epsilon (ε) isotypes. Each L chain may have a light chain variable region (VL) at the N-terminus and a constant domain at its other end, where the VL may correspond to the VH, and the light chain constant region (CL) may correspond to the first constant domain (CH1) of the heavy chain.Certain amino acid residues can be considered to form an interface between the light and heavy chain variable domains. VH and VL can pair with each other to form a single antigen-binding site. L chains from any vertebrate species can be classified into one of two different types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be classified into different classes or isotypes depending on the amino acid sequence of the constant domain of the heavy chain constant region (CH). There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, e.g., IgG1, IgG2, IgG3, and / or IgG4, and IgM, with heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), respectively.

[0170] In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., Siglec). In this application, an antigen-binding fragment can include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0171] In this application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As described above, an intact antibody can be digested with papain. Digestion of an antibody with papain produces two identical antigen-binding fragments, a "Fab" fragment and a remaining "Fc" fragment (i.e., the Fc region). A Fab fragment can consist of one complete L chain with the variable region of the heavy chain and the first constant region (CH1) of the H chain (VH).

[0172] In this application, the term "F(ab)2" generally refers to an antigen-binding fragment of an antibody. For example, F(ab)2 may be linked by two Fab fragments.

[0173] In this application, the term "Fab" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than a Fab fragment. For example, a Fab' fragment may comprise the entire light chain, the entire heavy chain variable region, and all or a portion of the first and second constant regions of the heavy chain. For example, a Fab' fragment may also include some or all of amino acid residues 220-330 of the heavy chain.

[0174] In this application, the term "F(ab')2" generally refers to an antibody fragment generated by digestion of an intact antibody with pepsin. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigens.

[0175] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody that contains all or part of the heavy and light chain variable regions and lacks the heavy and light chain constant regions. The heavy and light chain variable regions may, for example, contain CDRs. For example, the Fv fragment contains all or part of the amino-terminal variable regions of about 110 amino acids of the heavy and light chains.

[0176] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker, such as a short flexible polypeptide linker) and can be expressed as a single polypeptide chain, where the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv may have the VL and VH variable regions listed in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and may comprise a VL-linker-VH or a VH-linker-VL.

[0177] In this application, the term "di-scFv" generally refers to a divalent scFv, e.g., a molecule in which two scFv molecules are conjugated via a short linker.

[0178] In this application, the term "dAb" generally refers to an antigen-binding fragment consisting of a VH or VL domain composition. See, e.g., Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), see Holt et al., Trends Biotechnol., 2003,21(11):484-490.

[0179] In the present application, the term "VHH" generally refers to an antibody comprising a variable antigen-binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs can also be referred to as nanobodies (Nbs).

[0180] As used herein, the term "monoclonal antibody" generally refers to a preparation of antibody molecules consisting of a single molecule. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which generally have different antibodies directed against different determinants), each monoclonal antibody can be directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" can indicate the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies as used herein can be produced in hybridoma cells or by recombinant DNA methods.

[0181] In this application, the term "chimeric antibody" generally refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent (the "parent antibody"), and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to provoke a harmful immune response in a human individual compared to the parent (e.g., mouse-derived) antibody.

[0182] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDRs of a non-human antibody (e.g., a murine antibody) are replaced with the corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDRs are also acceptable as long as the antibody still retains its ability to bind to a specific antigen. A humanized antibody may optionally comprise at least a portion of a constant region of a human immunoglobulin. A "humanized antibody" may retain the same antigen specificity as the original antibody. "Humanized" forms of non-human antibodies (e.g., a murine antibody) may minimally comprise chimeric antibodies derived from non-human immunoglobulin sequences. In some cases, residues of the CDRs of a human immunoglobulin (receptor antibody) may be replaced with residues of the CDRs from a non-human species (donor antibody) (e.g., mouse, rat, rabbit or non-human primate) having the desired properties, affinities, and / or capacities. In some cases, residues of the FR regions of a human immunoglobulin may be replaced with the corresponding non-human residues. Furthermore, humanized antibodies may comprise amino acid modifications that are not present in the receptor antibody or in the donor antibody. These modifications can be made to further improve antibody properties, such as binding affinity.

[0183] In this application, the term "fully human antibody" generally refers to an antibody that comprises only human immunoglobulin protein sequences. A fully human antibody may contain mouse glycochains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody," "mouse antibody," or "rat antibody" refers to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in human transgenic animals with human immunoglobulin germline sequences, or by phage display or other molecular biology methods. Exemplary techniques that can be used to produce antibodies are known in the art.

[0184] In this application, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or backbone portion that allows the antigen-binding portion to assume a conformation that facilitates binding of the antigen-binding protein to the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb), immune complexes, antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, so long as they exhibit the desired antigen-binding activity. An "isolated antigen-binding protein" in this application may comprise an antigen-binding portion and, optionally, a scaffold or backbone portion that allows the antigen-binding portion to assume a conformation that facilitates binding of the antigen-binding portion to the antigen.

[0185] In this application, the terms "polypeptide molecule" and "polypeptide" and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two moieties covalently linked to each other. Each moiety can be a polypeptide with different properties. The properties can be biological properties, such as in vitro activity or in vivo activity. The properties can also be simple chemical or physical properties, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties can be directly linked by a single peptide bond or a peptide linker.

[0186] As used herein, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides, or ribonucleotides, or analogs isolated from their natural environment or synthesized.

[0187] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and expressed. Expression of the genetic material elements carried by the vector in a host cell can be achieved by transformation, transduction or transfection of the vector into the host cell. The vector may contain multiple elements for controlling expression. Alternatively, the vector may comprise an origin of replication. The vector may also contain components that facilitate the entry of the vector into a cell.

[0188] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject-derived plasmid or vector, including a nucleic acid molecule of the present application or a vector of the present application. A cell can include the progeny of a single cell. The progeny may not necessarily be identical (in morphology or genome to total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. The cell can include a cell that has been transfected in vitro with a vector of the present application.

[0189] As used herein, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., via the covalent attachment of a bridging molecule) of an antibody or antigen-binding fragment thereof with another reagent (e.g., chemotherapeutic drugs, radioactive elements, cytostatic agents, and cytotoxic agents) that is capable of delivering the other reagent to a target cell (e.g., a tumor cell) via specific binding of the antibody or antigen-binding fragment thereof to an antigen on the target cell.

[0190] In the present application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein of the present application, the nucleic acid molecule of the present application, the vector of the present application, and / or the cell of the present application, and optionally, a pharma- ceutical acceptable adjuvant. In addition, the pharmaceutical composition may also comprise suitable preparations, such as one or more (pharma- ceutical effective) vehicles. Acceptable components of these compositions may be non-toxic to recipients at the dosages and concentrations used. The pharmaceutical composition of the present application includes, but is not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0191] In this application, the term "pharmaceutically acceptable vehicle" generally refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed at the dosage and concentration used. A physiologically acceptable carrier may comprise a suitable substance. A pharmaceutically acceptable carrier is generally not of the same class as a vector used to insert a nucleic acid in genetic engineering.

[0192] In this application, the term "solid tumor" generally refers to a palpable tumor that is detectable by clinical examination (e.g., X-ray, CT scan, B-ultrasound, or palpation, etc.). A tumor may include a neoplasm or a solid lesion formed by abnormal cell proliferation.

[0193] In this application, the term "colon cancer" generally refers to tumors that occur or originate in the colonic region of the digestive tract.For example, colon cancer can occur or originate at the junction of the rectum and the sigmoid colon.For example, colon cancer can be cancer that occurs or originates in the colonic region of the digestive tract.

[0194] In the present application, the term "direct linkage" can be contrasted with the term "indirect linkage" and generally means a direct bond. For example, direct linkage can be a state in which substances are directly linked to each other without using a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirect linkage" generally refers to a state in which substances are not regulated and linked to each other. For example, indirect linkage can be a state in which substances are linked to each other via a spacer. For example, in the isolated antigen-binding protein of the present application, the C-terminus of L-FR1 and the N-terminus of LCDR1 can be directly or indirectly linked.

[0195] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to that target with higher affinity, avidity, more readily, and / or for longer than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In some embodiments, specific binding can include, but does not have to be, exclusive binding.

[0196] As used herein, the term "subject" generally refers to a human or a non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0197] In this application, the term "tumor" generally refers to any new pathological tissue hyperplasia. Tumor cells may spread locally or via the bloodstream and lymphatic system to other parts of the body. In this application, tumors may include benign and malignant tumors. In this application, tumors may include solid tumors and / or hematological tumors. In this application, tumors may include cancers. In this application, examples of tumors include, but are not limited to, colon cancer.

[0198] In this application, references to proteins, polypeptides and / or amino acid sequences should be understood to encompass at least the following scope: variants or homologs having the same or similar function as the protein or polypeptide.

[0199] In the present application, a variant can be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to Siglec). For example, a functional variant can comprise a protein or polypeptide that has been altered by at least one, e.g., 1-30, 1-20 or 1-10, even e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. A functional variant can substantially retain the biological properties of the protein or polypeptide prior to alteration (e.g., substitution, deletion or addition). For example, a functional variant can retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide prior to alteration. For example, the substitutions can be conservative substitutions. For example, a variant can also be a polypeptide including a functionally active fragment of a protein resulting from processing and / or modification that occurs within a cell, including, but not limited to, a functionally active fragment of the protein.

[0200] In the present application, a homolog can be a protein or polypeptide that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the amino acid sequence of a protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to Siglec).

[0201] In this application, homology generally refers to the similarity, similarity or relationship between two or more sequences. "Percentage of sequence homology" refers to the following: two sequences to be aligned are compared over a comparison window, the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in the two sequences is determined to obtain the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to obtain the percentage of sequence homology. Alignment to determine the percentage of sequence homology can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared or over a region of the target sequence. Homology can also be determined by the following methods: FASTA and BLAST. The FASTA algorithm is described in WR Pearson and DJ Lipman "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ Lipman and WR Pearson "Rapid and Sensitive Protein Similarity Searches", Science, 227: 1435-1441, 1989. The BLAST algorithm is described in S. Altschul, W. Gish, W. Miller, EW Myers and D. Lipman, A Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403-410, 1990.

[0202] In this application, the term "comprises" or other forms of this term refer to comprising, including, containing, or including. In some cases, this term also refers to the meanings "is" and "consisting of."

[0203] In this application, the term "about" generally refers to a range of 0.5% to 10% above or below a particular value, for example, 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% above or below a particular value.

[0204] Detailed Description of the Invention In one aspect, the present application provides an isolated antigen binding protein.

[0205] The CDR of an antibody, also known as the complementarity determining region, is a part of the variable region. The amino acid residues of this region may contact the antigen or antigen epitope. The CDR of an antibody can be identified by various coding systems known in the art. For example, see www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR using different coding systems based on the sequence and structure of the antibody. The CDR may be different in different coding systems. In the present application, CDR covers the CDR sequence obtained by dividing according to any CDR division manner; variants thereof comprising the amino acid sequence of the CDR having one or more amino acid substitutions, deletions, and / or additions, for example, 1-30, 1-20, or 1-10, even for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions, are also contemplated; and homologs thereof, which may be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the CDR are also contemplated. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the Chothia numbering scheme.

[0206] In one aspect, the present application provides an isolated antigen binding protein comprising an HCDR3 comprising an amino acid sequence as set forth in AIGSSWYSDAFDL (SEQ ID NO: 1). For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the isolated antigen binding protein may comprise the HCDR3 of the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the CDRs are determined according to Chothia rules.

[0207] In the present application, the isolated antigen-binding protein may further comprise HCDR2. For example, HCDR2 may comprise a sequence as shown in X1GGGX2X3, where X1, X2, and X3 can each be independently selected from any amino acid, such as a natural amino acid. For example, HCDR2 may comprise a sequence as shown in X1GGGX2X3 (SEQ ID NO: 33), where X1 can be S, T, W, or Y, X2 can be E, G, or V, and X3 can be S or Y. In some cases, compared to the amino acid sequence as shown in SEQ ID NO: 2, HCDR2 may comprise at least one amino acid substitution at a position selected from the group consisting of amino acid substitutions at X1, X2, and X3. For example, HCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 33. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability.

[0208] For example, the HCDR2 may comprise an amino acid sequence as shown in SGGGGS (SEQ ID NO:2), TGGGES (SEQ ID NO:3), YGGGGS (SEQ ID NO:4), WGGGGS (SEQ ID NO:5), or SGGGVY (SEQ ID NO:6). For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise HCDR2 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the isolated antigen-binding protein may comprise HCDR2 of the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0209] In the present application, the isolated antigen binding protein may further comprise HCDR1. For example, HCDR1 may comprise a sequence as shown in GFTX4SSY, where X4 may be selected from any amino acid, such as a natural amino acid. For example, HCDR1 may comprise a sequence as shown in GFTX4SSY (SEQ ID NO: 34), where X4 may be F or S. In some cases, HCDR1 may comprise an amino acid substitution at least in X4, compared to the amino acid sequence as shown in SEQ ID NO: 7. For example, HCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 34. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0210] For example, HCDR1 may comprise an amino acid sequence as shown in GFTFSSY (SEQ ID NO: 7) or GFTSSSY (SEQ ID NO: 8). For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the isolated antigen binding protein may comprise HCDR1 of the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the CDRs are determined according to Chothia rules.

[0211] In the present application, the isolated antigen binding protein may comprise HCDR3, HCDR2, and HCDR1. For example, HCDR3 may comprise a sequence as shown in SEQ ID NO: 1, HCDR2 may comprise a sequence as shown in X1GGGX2X3, where X1, X2, and X3 can each independently be selected from any amino acid, such as a natural amino acid, and HCDR1 may comprise a sequence as shown in GFTX4SSY, where X4 can be selected from any amino acid, such as a natural amino acid. For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO: 1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO: 33; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO: 34. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0212] For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:2; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:7. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of Ab0 or Ab9 of the present application. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of Ab0 or Ab9 of the present application. For example, the CDRs are determined according to Chothia rules.

[0213] For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:2; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:8. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of Ab2 of the present application. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of Ab2 of the present application. For example, the CDRs are determined according to Chothia rules.

[0214] For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:3; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:7. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of Ab3, Ab13, or Ab15 of the present application. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of Ab3, Ab13, or Ab15 of the present application. For example, the CDRs are determined according to Chothia rules.

[0215] For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:4; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:7. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of Ab5 of the present application. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of Ab5 of the present application. For example, the CDRs are determined according to Chothia rules.

[0216] For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:5; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:7. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of Ab6 of the present application. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of Ab6 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0217] For example, the HCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:6; the HCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:7. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of Ab7 of the present application. For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of Ab7 of the present application. For example, the CDRs are determined according to Chothia rules.

[0218] In the present application, the isolated antigen binding protein may comprise an LCDR3 that may comprise an amino acid sequence as shown in QQSYSIPYT (SEQ ID NO: 9). For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the LCDR3 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the isolated antigen binding protein may comprise the LCDR3 of the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the CDR is determined according to the Chothia rules.

[0219] In the present application, the isolated antigen-binding protein may further comprise LCDR2. For example, LCDR2 may comprise a sequence as shown in VASX5X6X7X8, where X5, X6, X7, and X8 can each independently be selected from any amino acid, such as a natural amino acid. For example, LCDR2 may comprise a sequence as shown in VASX5X6X7X8 (SEQ ID NO: 35), where X5 can be F or S, X6 can be E, I, or L, X7 can be H or Q, and X8 can be R or S. In some cases, compared to the amino acid sequence as shown in SEQ ID NO: 10, LCDR2 may comprise at least one amino acid substitution at a position selected from the group consisting of amino acid substitutions at X5, X6, X7, and X8. For example, LCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 35. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability.

[0220] For example, LCDR2 may comprise an amino acid sequence as shown in VASSLQS (SEQ ID NO: 10), VASFEHR (SEQ ID NO: 11), or VASYIHS (SEQ ID NO: 12). For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise LCDR2 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the isolated antigen binding protein may comprise LCDR2 of the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the CDRs are determined according to Chothia rules.

[0221] In the present application, the isolated antigen-binding protein may further comprise LCDR1. For example, LCDR1 may comprise a sequence as shown in RASQDISX9WLA, where X9 may be selected from any amino acid, such as a natural amino acid. For example, LCDR1 may comprise a sequence as shown in RASQDISX9WLA (SEQ ID NO: 36), where X9 may be D or S. In some cases, LCDR1 may comprise an amino acid substitution at least in X9, compared to the amino acid sequence as shown in SEQ ID NO: 13. For example, LCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 36. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability.

[0222] For example, LCDR1 may comprise an amino acid sequence as shown in RASQDISSWLA (SEQ ID NO: 13) or RASQDISDWLA (SEQ ID NO: 14). For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise LCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the isolated antigen binding protein may comprise LCDR1 of the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application. For example, the CDRs are determined according to Chothia rules.

[0223] In the present application, the isolated antigen binding protein may comprise LCDR3, LCDR2, and LCDR1. For example, LCDR3 may comprise a sequence as shown in SEQ ID NO: 9, LCDR2 may comprise a sequence as shown in VASX5X6X7X8, where X5, X6, X7, and X8 can each independently be selected from any amino acid, such as a natural amino acid, and LCDR1 may comprise a sequence as shown in RASQDISX9WLA, where X9 can be selected from any amino acid, such as a natural amino acid. For example, LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO: 9; LCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO: 35; LCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO: 36. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0224] For example, the LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of Ab0, Ab2, Ab3, Ab5, or Ab7 of the present application. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of the light chain variable region of Ab0, Ab2, Ab3, Ab5, or Ab7 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0225] For example, the LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:14. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of Ab9 of the present application. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of the light chain variable region of Ab9 of the present application. For example, the CDRs are determined according to Chothia rules.

[0226] For example, the LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:11; the LCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of Ab13 of the present application. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of the light chain variable region of Ab13 of the present application. For example, the CDRs are determined according to Chothia rules.

[0227] For example, the LCDR3 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:12; the LCDR1 of the isolated antigen binding protein may comprise an amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of Ab15 of the present application. For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of the light chain variable region of Ab15 of the present application. For example, the CDRs are determined according to Chothia rules.

[0228] In the present application, the isolated antigen binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1. For example, HCDR3 may comprise a sequence as shown in SEQ ID NO: 1; HCDR2 may comprise a sequence as shown in X1GGGX2X3, where X1, X2, and X3 can each independently be selected from any amino acid, such as a natural amino acid; HCDR1 may comprise a sequence as shown in GFTX4SSY, where X4 can each independently be selected from any amino acid, such as a natural amino acid; LCDR3 may comprise a sequence as shown in SEQ ID NO: 9; LCDR2 may comprise a sequence as shown in VASX5X6X7X8, where X5, X6, X7, and X8 can each independently be selected from any amino acid, such as a natural amino acid; LCDR1 may comprise a sequence as shown in RASQDISX9WLA, where X9 can each independently be selected from any amino acid, such as a natural amino acid. For example, HCDR3 may comprise a sequence as shown in SEQ ID NO:1; HCDR2 may comprise a sequence as shown in X1GGGX2X3 (SEQ ID NO:33), where X1 may be S, T, W or Y, X2 may be E, G or V, and X3 may be S or Y; HCDR1 may comprise a sequence as shown in GFTX4SSY (SEQ ID NO:34), where X4 may be F or S; LCDR3 may comprise a sequence as shown in SEQ ID NO:9; LCDR2 may comprise a sequence as shown in VASX5X6X7X8 (SEQ ID NO:35), where X5 may be F, S or Y, X6 may be E, I or L, X7 may be H or Q, and X8 may be R or S; LCDR3 may comprise a sequence as shown in RASQDISX9WLA (SEQ ID NO:36), where X9 may be D or S.In some cases, compared to the amino acid sequence as shown in SEQ ID NO:2, HCDR2 may comprise at least one amino acid substitution at a position selected from the group consisting of amino acid substitutions at X1, X2 and X3; compared to the amino acid sequence as shown in SEQ ID NO:7, HCDR1 may comprise at least an amino acid substitution at X4; compared to the amino acid sequence as shown in SEQ ID NO:10, LCDR2 may comprise at least one amino acid substitution at a position selected from the group consisting of amino acid substitutions at X5, X6, X7 and X8; compared to the amino acid sequence as shown in SEQ ID NO:13, LCDR1 may comprise at least an amino acid substitution at X9. For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:33; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:34; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:35; and the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:36. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0229] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:2; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab0 of the present application. For example, an isolated antigen binding protein can comprise HCDR3, HCDR2, HCDR1 of the heavy chain variable region and LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab0 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0230] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:2; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:8; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab2 of the present application. For example, an isolated antigen binding protein can comprise the HCDR3, HCDR2, and HCDR1 of the heavy chain variable region and the LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab2 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0231] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:3; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab3 of the present application. For example, an isolated antigen binding protein can comprise the HCDR3, HCDR2, and HCDR1 of the heavy chain variable region and the LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab3 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0232] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:4; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab5 of the present application. For example, an isolated antigen binding protein can comprise the HCDR3, HCDR2, and HCDR1 of the heavy chain variable region and the LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab5 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0233] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:5; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab6 of the present application. For example, an isolated antigen binding protein can comprise HCDR3, HCDR2, HCDR1 of the heavy chain variable region and LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab6 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0234] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:6; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab7 of the present application. For example, an isolated antigen binding protein can comprise the HCDR3, HCDR2, and HCDR1 of the heavy chain variable region and the LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab7 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0235] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:2; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:10; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:14. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab9 of the present application. For example, an isolated antigen binding protein can comprise the heavy chain variable region HCDR3, HCDR2, HCDR1, and the light chain variable region LCDR3, LCDR2, and LCDR1 of Ab9 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0236] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:3; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:11; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as set forth in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab13 of the present application. For example, an isolated antigen binding protein can comprise HCDR3, HCDR2, HCDR1 of the heavy chain variable region and LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab13 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0237] For example, the HCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:1; the HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:3; the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:7; the LCDR3 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:9; the LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:12; the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence as shown in SEQ ID NO:13. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab15 of the present application. For example, an isolated antigen binding protein can comprise HCDR3, HCDR2, HCDR1 of the heavy chain variable region and LCDR3, LCDR2, and LCDR1 of the light chain variable region of Ab15 of the present application. For example, the CDRs are determined according to the Chothia rules.

[0238] In the present application, the isolated antigen binding protein may comprise H-FR1, the C-terminus of HCDR1 may be directly or indirectly linked to the N-terminus of HCDR1, and H-FR1 may comprise an amino acid sequence as set forth in SEQ ID NO:15.

[0239] In the present application, the isolated antigen binding protein comprises H-FR2, which is located between HCDR1 and HCDR2, and which may comprise an amino acid sequence as set forth in SEQ ID NO:16.

[0240] In the present application, the isolated antigen binding protein may comprise H-FR3, which is located between HCDR2 and HCDR3, and which may comprise an amino acid sequence as set forth in SEQ ID NO:17.

[0241] In the present application, the isolated antigen binding protein may comprise H-FR4, the N-terminus of H-FR4 may be linked to the C-terminus of HCDR3, and H-FR4 may comprise an amino acid sequence as set forth in SEQ ID NO:18.

[0242] In the present application, an antigen binding protein can comprise H-FR1, H-FR2, H-FR3, and H-FR4. For example, H-FR1, H-FR2, H-FR3, and H-FR4 of an isolated antigen binding protein can comprise the amino acid sequences as shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively.

[0243] In the present application, the isolated antigen binding protein may comprise L-FR1, the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1, and L-FR1 may comprise an amino acid sequence as shown in SEQ ID NO:19.

[0244] In the present application, the isolated antigen binding protein may comprise L-FR2, which is located between LCDR1 and LCDR2, and which may comprise an amino acid sequence as shown in SEQ ID NO:20.

[0245] In the present application, the isolated antigen binding protein comprises L-FR3, which is located between LCDR2 and LCDR3, and which may comprise the amino acid sequence as shown in SEQ ID NO:21.

[0246] In the present application, the isolated antigen binding protein may comprise L-FR4, the N-terminus of L-FR4 may be directly or indirectly linked to the C-terminus of LCDR3, and L-FR4 may comprise an amino acid sequence as shown in SEQ ID NO:22.

[0247] In the present application, the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3, and L-FR4. For example, the L-FR1, L-FR2, L-FR3, and L-FR4 of the isolated antigen binding protein may comprise the amino acid sequences as shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively.

[0248] In the present application, the isolated antigen binding protein may comprise a VH, which may comprise an amino acid sequence as set forth in SEQ ID NO: 37. For example, the VH may comprise [ka] wherein X1 can be S, T, W, or Y, X2 can be E, G, or V, X3 can be S or Y, and X4 can be F or S. Optionally, compared to the amino acid sequence as set forth in SEQ ID NO: 23, VH can comprise at least one amino acid substitution at a position selected from the group consisting of amino acid substitutions at X1, X2, X3, and X4. For example, the isolated antigen binding protein of the present application can have Siglec-15 binding ability.

[0249] In the present application, the antigen binding protein may comprise a VH, which may comprise an amino acid sequence as set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application.

[0250] In the present application, the isolated antigen binding protein may comprise a VL, which may comprise an amino acid sequence as set forth in SEQ ID NO: 38. For example, the VL may comprise [ka] wherein X5 can be F, S, or Y, X6 can be E, I, or L, X7 can be H or Q, X8 can be R or S, and X9 can be D or S. In some cases, compared to the amino acid sequence as set forth in SEQ ID NO: 29, the VH may comprise at least one amino acid substitution at a position selected from the group consisting of amino acid substitutions at X5, X6, X7, X8, and X9. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability.

[0251] In the present application, the antigen binding protein may comprise a VL, which may comprise an amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab7, Ab9, Ab13, or Ab15 of the present application.

[0252] In the present application, the isolated antigen-binding protein may comprise a VH and a VL. For example, the VH may comprise an amino acid sequence as shown in SEQ ID NO: 37, and the VL may comprise an amino acid sequence as shown in SEQ ID NO: 38. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability.

[0253] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 23, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab0 of the present application.

[0254] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 24, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the VH and VL of Ab2 of the present application.

[0255] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 25, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen binding protein may comprise the VH and VL of Ab3 of the present application.

[0256] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 26, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab5 of the present application.

[0257] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 27, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab6 of the present application.

[0258] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 28, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab7 of the present application.

[0259] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 23, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 30. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab9 of the present application.

[0260] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 25, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 31. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab13 of the present application.

[0261] For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 25, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 32. For example, the isolated antigen-binding protein of the present application may have Siglec-15 binding ability. For example, the isolated antigen-binding protein may comprise the VH and VL of Ab15 of the present application.

[0262] In the present application, the isolated antigen-binding protein may comprise at least one CDR of any of the VHs of the present application. In the present application, the isolated antigen-binding protein may comprise at least one CDR of any of the VLs of the present application. The CDRs may be determined according to any classification scheme, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia comprehensive considerations.

[0263] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the VH of the present application, and the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 37. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0264] In the present application, the antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 of the VH of the present application, and the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0265] In the present application, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of the VL of the present application, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 38. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0266] In the present application, the antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 of the VL of the present application, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. For example, the isolated antigen binding protein of the present application may have Siglec-15 binding ability.

[0267] In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG, IgA, IgD, IgE and / or IgM heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In some embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1, IgG2, IgG3 and / or IgG4 heavy chain constant region. In some embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

[0268] In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region. The antibody light chain constant region may be derived from a human Igκ constant region.

[0269] In this application, an isolated antigen binding protein may comprise an antibody or an antigen binding fragment thereof.

[0270] In some embodiments, the antigen-binding fragment may include a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv, a VHH, and / or a dAb.

[0271] In some embodiments, the antibody may comprise a monoclonal antibody. In some embodiments, the antibody may comprise a murine antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0272] For example, the VH of the chimeric antibody can comprise the amino acid sequence as set forth in SEQ ID NO:23, and the VL of the chimeric antibody can comprise the amino acid sequence as set forth in SEQ ID NO:29.

[0273] In some embodiments, the VH of the humanized antibody may comprise an amino acid sequence as set forth in SEQ ID NO:37, and the VL of the humanized antibody may comprise an amino acid sequence as set forth in SEQ ID NO:38. For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO:23, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO:29. For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO:24, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO:29. For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO:25, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO:29. For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO:26, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO:29. For example, the VH may comprise an amino acid sequence as set forth in SEQ ID NO:27, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO:29. For example, the VH may comprise the amino acid sequence as set forth in SEQ ID NO:28 and the VL may comprise the amino acid sequence as set forth in SEQ ID NO:29. For example, the VH may comprise the amino acid sequence as set forth in SEQ ID NO:23 and the VL may comprise the amino acid sequence as set forth in SEQ ID NO:30. For example, the VH may comprise the amino acid sequence as set forth in SEQ ID NO:25 and the VL may comprise the amino acid sequence as set forth in SEQ ID NO:31. For example, the VH may comprise the amino acid sequence as set forth in SEQ ID NO:25 and the VL may comprise the amino acid sequence as set forth in SEQ ID NO:32.

[0274] In some embodiments, the antibody comprises a light chain constant region. For example, the light chain constant region can be a light chain constant region derived from a human antibody; for example, the light chain constant region can comprise the amino acid sequence as set forth in SEQ ID NO:40.

[0275] In some embodiments, the antibody comprises a heavy chain constant region. For example, the heavy chain constant region can be a heavy chain constant region derived from a human antibody; for example, the heavy chain constant region can comprise the amino acid sequence as set forth in SEQ ID NO:39.

[0276] It should further be noted that the isolated antigen-binding protein of the present application may comprise a heavy and / or light chain sequence having one or more conservative sequence modifications. "Conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding protein of the present application by standard techniques known in the art, such as point mutation and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDRs of the isolated antigen binding proteins of the present application can be substituted with other amino acid residues of the same side chain group. One of skill in the art will recognize that some conservative sequence modifications do not impair antigen binding.For example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0277] Siglec-15 antigen binding proteins of the present application can be identified, screened, or characterized by a variety of assays known in the art.

[0278] For example, the antigen-binding proteins or fusion proteins of the present application can be tested for antigen-binding activity by known methods, such as enzyme-linked immunosorbent assay (ELISA), immunoblot (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, and immunofluorescence.

[0279] In the present application, an isolated antigen binding protein can specifically bind to a Siglec or a functionally active fragment thereof, such as Siglec-15 or a functionally active fragment thereof.

[0280] In some embodiments, Siglec-15 or a functionally active fragment thereof can be full-length Siglec-15 or a functionally active fragment thereof, or a fragment that exerts Siglec-15 functional activity, e.g., Siglec-15 functional activity can be the ability to activate a downstream pathway of Siglec-15. In some embodiments, Siglec-15 or a functionally active fragment thereof can be an isolated Siglec-15 or a functionally active fragment thereof, or a mixture of various forms of Siglec-15 or a functionally active fragment thereof. For example, Siglec-15 or a functionally active fragment thereof can be human Siglec-15 or a functionally active fragment thereof, mouse Siglec-15 or a functionally active fragment thereof, or monkey Siglec-15 or a functionally active fragment thereof; for example, Siglec-15 or a functionally active fragment thereof can be Siglec-15 or a functionally active fragment thereof expressed on the surface of a cell; for example, Siglec-15 or a functionally active fragment thereof can be Siglec-15 or a functionally active fragment thereof bound to a solid phase medium.

[0281] In some embodiments, the isolated antigen binding protein can have its affinity measured by a molecular interaction analyzer. For example, the isolated antigen binding protein can have a K of 1.56E-10 M or less. D For example, the isolated antigen binding protein can bind to the Siglec-15 protein with a K value of 1.56E-10 M or less, 1.5E-10 M or less, 1.3E-10 M or less, 1.1E-10 M or less, 1.0E-10 M or less, 9.0E-11 M or less, 8.0E-11 M or less, 7.0E-11 M or less, 6.0E-11 M or less, 5.0E-11 M or less, 4.0E-11 M or less, 3.0E-11 M or less, 2.0E-11 M or less, 1.0E-11 M or less, 9.0E-12 M or less, 8.0E-12 M or less, 7.0E-12 M or less, 6.0E-12 M or less, or 5.0E-12 M or less. DThe antibody can bind to the Siglec-15 protein at a concentration of 0.015 or lower.

[0282] In some embodiments, the isolated antigen binding protein of the present application can be expressed in higher amounts. For example, the isolated antigen binding protein of the present application can be expressed in an amount of 50 mg / L or more. For example, the isolated antigen binding protein of the present application can be expressed in an amount of 50 mg / L or more, 53 mg / L or more, 55 mg / L or more, 60 mg / L or more, 70 mg / L or more, 80 mg / L or more, 90 mg / L or more, 100 mg / L or more, 130 mg / L or more, 140 mg / L or more, 150 mg / L or more, 160 mg / L or more, 200 mg / L or more, 210 mg / L or more, 220 mg / L or more, or 230 mg / L or more.

[0283] In some embodiments, the isolated antigen-binding protein of the present application can be measured for its binding activity to cell surface Siglec-15 protein by FACS. For example, it can be cell surface human Siglec-15 protein, cell surface monkey Siglec-15 protein, and / or cell surface mouse Siglec-15 protein. For example, the isolated antigen-binding protein of the present application has an EC of about 3.25 nM or less. 50 For example, the isolated antigen binding protein of the present application can bind to Siglec-15 or a functional fragment thereof with an EC value of about 3.25 nM or less, about 3.0 nM or less, about 2.0 nM or less, about 1.9 nM or less, about 1.8 nM or less, about 1.7 nM or less, about 1.6 nM or less, about 1.5 nM or less, about 1.4 nM or less, about 1.3 nM or less, about 1.2 nM or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, or about 0.1 nM or less. 50 The present invention is capable of binding to Siglec-15 or a functional fragment thereof at a value.

[0284] In some embodiments, the isolated antigen binding proteins of the present application can affect the proliferation capacity of immune cells. For example, the isolated antigen binding proteins of the present application can affect the function of the immune system; for example, the isolated antigen binding proteins of the present application can affect the proportion of immune cell subpopulations and / or the number of immune cells. For example, immune cells can include lymphocytes, peripheral blood lymphocytes, peripheral blood mononuclear cells, PBMCs, T cells, B cells, and / or NK cells. For example, the isolated antigen binding proteins of the present application can increase the proportion of immune cell subpopulations; for example, the isolated antigen binding proteins of the present application can increase the proportion of immune cell subpopulations; for example, the isolated antigen binding proteins of the present application can increase the proliferation capacity of immune cells ... + For example, the isolated antigen binding proteins of the present application can enhance the proportion of CD4 + For example, the isolated antigen-binding protein of the present application can increase the proportion of CD8 T cells in peripheral blood lymphocytes. + For example, the isolated antigen-binding proteins of the present application can enhance the proportion of CD4 T cells in peripheral blood lymphocytes. + For example, the isolated antigen-binding protein of the present application can enhance the proportion of CD8 + The number of T cells can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to a group administered only a blank control. For example, the isolated antigen binding protein of the present application can be a CD4 + The number of T cells can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to a group administered only a blank control.

[0285] In some embodiments, the isolated antigen binding protein of the present application can reverse the suppressive effect of Siglec-15 protein on immune system function. For example, when Siglec-15 protein is administered in vivo or in vitro and / or Siglec-15 protein is overexpressed in vivo or in vitro, Siglec-15 protein can suppress immune system function, e.g., Siglec-15 protein can affect the proportion of immune cell subpopulations, suppress immune cell proliferation, and / or reduce the number of immune cells. For example, when immune system function is suppressed by Siglec-15 protein, the isolated antigen binding protein of the present application can improve, reverse, restore, and / or activate immune system function. For example, after Siglec-15 protein suppresses immune system function and / or immune cell number, the isolated antigen binding protein of the present application can suppress CD8 + The number of T cells can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to a group administered only a blank control. For example, after the Siglec-15 protein has suppressed immune system function and / or immune cell numbers, the isolated antigen binding protein of the present application can inhibit CD4 + The number of T cells can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to a group administered only a blank control.

[0286] In some embodiments, the isolated antigen binding protein of the present disclosure can suppress tumor growth. For example, the isolated antigen binding protein has the ability to suppress the growth of a tumor, such as a colon cancer, with a tumor volume inhibition rate of about 23% or more. For example, the isolated antigen binding protein has the ability to suppress tumor growth with a tumor volume inhibition rate of about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 40% or more, or about 50% or more, compared to a group administered only a blank control.

[0287] In some embodiments, the isolated antigen binding proteins of the present application can affect the ability of cytokine secretion. For example, the secretion of the cytokines of the present application can include in vivo, ex vivo, and / or in vitro secretion. For example, the secretion of the cytokines of the present application can include cytokine secretion induced by lipopolysaccharide. For example, the cytokine secretion induced by lipopolysaccharide can refer to an increase in the amount of cytokine secreted in vivo and / or in vitro, an increase in the cytokine concentration in the culture medium, and / or an increase in the cytokine concentration in the peripheral blood after administration of lipopolysaccharide in vivo and / or in vitro. For example, administering the antigen binding protein of the present application and then lipopolysaccharide to a subject results in an increase in the amount of cytokine in the subject compared to administration of lipopolysaccharide alone or lipopolysaccharide followed by a blank. For example, compared to administration of lipopolysaccharide alone or bran followed by lipopolysaccharide, the antigen binding proteins of the present application can increase cytokine secretion by greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 7%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater than about 150%, greater than about 200%, greater than about 250%, greater than about 300%, greater than about 350%, greater than about 400%. For example, cytokines of the present application can include factors associated with an inflammatory response or inflammatory factors such as the proinflammatory factors TNF-α and / or IL-6.For example, compared to administration of lipopolysaccharide alone or bran followed by lipopolysaccharide, the antigen binding proteins of the present application can increase TNF-α secretion by greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 7%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater than about 150%, greater than about 200%, greater than about 250%, greater than about 300%, greater than about 350%, greater than about 400%. For example, compared to administration of lipopolysaccharide alone or bran followed by lipopolysaccharide, the antigen binding proteins of the present application can increase IL-6 secretion by greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 7%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater than about 150%, greater than about 200%, greater than about 250%, greater than about 300%, greater than about 350%, greater than about 400%.

[0288] In one aspect, the present application provides polypeptide molecules, nucleic acid molecules, vectors, immunoconjugates, cells, and pharmaceutical compositions.

[0289] In another aspect, the present application provides a polypeptide molecule that can comprise an isolated antigen binding protein of the present application.

[0290] In some embodiments, the polypeptide molecule may comprise a fusion protein. In some embodiments, the polypeptide molecule may be a fusion protein. In some embodiments, the polypeptide molecule of the present application may comprise structures other than amino acids, for example, the polypeptide molecule of the present application may comprise nucleic acids, polysaccharides, lipids, small molecules, and any combination of the above.

[0291] In another aspect, the present application provides an isolated nucleic acid molecule capable of encoding the isolated antigen binding protein of the present application and / or the polypeptide of the present application. For example, the nucleic acid molecule can be produced or synthesized by the following methods: (i) in vitro amplification, e.g., polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii) purification, e.g., digestion and gel electrophoretic fractionation, or (iv) synthesis, e.g., chemical synthesis.

[0292] In another aspect, the present application provides a vector that may comprise the nucleic acid molecule of the present application. In addition, the vector may also comprise other genes, such as marker genes, that allow the selection of the vector under appropriate conditions in a suitable host cell. In addition, the vector may also comprise expression control elements that allow the proper expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. Expression of the genetic material elements carried by the vector in a host cell can be achieved by transformation, transduction, or transfection of the vector into the host cell. Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. In addition, the vector may also include components that aid the vector in entering a cell, including, but not limited to, viral particles, liposomes, or protein shells.

[0293] In another aspect, the present application also provides an immunoconjugate that may comprise the isolated antigen-binding protein of the present application and / or the polypeptide of the present application. In some embodiments, the isolated antigen-binding protein of the present application or a fragment thereof may be linked to another reagent, such as a chemotherapeutic drug, a toxin, an immunotherapeutic drug, an imaging probe, a spectroscopic probe, etc. The linkage may be formed through one or more covalent bonds or non-covalent interactions, and may include chelation. Various linkers that may be known in the art may be used to form the immunoconjugate. Furthermore, the immunoconjugate may be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate. The immunoconjugate may also comprise, for example, an antibody-drug ADC. In the ADC, the antibody and the therapeutic agent may be crosslinked by a linker, which may be a cleavable linker, for example, a linker that is enzymatically cleavable in vivo and / or in vitro, for example, a peptide linker, a disulfide linker, or a hydrazone linker.

[0294] In another aspect, the present application provides a cell that may comprise the isolated antigen binding protein of the present application, the polypeptide molecule of the present application, the immunoconjugate of the present application, the nucleic acid molecule of the present application, or the vector of the present application. In some embodiments, any host cell may comprise one or one type of nucleic acid molecule or vector of the present application. In some embodiments, any host cell may comprise a plurality (e.g., two or more) or a plurality (e.g., two or more) of nucleic acid molecules or vectors of the present application. For example, the vector of the present application can be introduced into a host cell, e.g., a eukaryotic cell, such as a cell derived from a plant, fungus, or yeast. In some embodiments, the cell can be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2 / 0 cell, a NS0 cell, or a myeloma cell. The vectors of the present application can be introduced into the host by methods known in the art, such as electroporation, lipofectin transfection, lipofectamine transfection, etc.

[0295] In another aspect, the present application also provides pharmaceutical compositions that may comprise the isolated antigen-binding proteins of the present application, the polypeptide molecules of the present application, the immunoconjugates of the present application, the nucleic acid molecules of the present application, the vectors of the present application, and / or the cells of the present application, and optionally a pharma- ceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions may further comprise suitable formulations of one or more (pharmaceutical effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the composition may be non-toxic to recipients at the dosages and concentrations used. Pharmaceutical compositions of the present invention may include, but are not limited to, liquids, frozen compositions, and lyophilized compositions.

[0296] In some embodiments, pharmaceutical compositions may also contain two or more active compounds, generally those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs may vary, for example, depending on the amount and type of antagonist present in the formulation, and the clinical parameters of the subject.

[0297] In some embodiments, pharma- ceutically acceptable vehicles may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and are generally safe, non-toxic, and , suitable for drug administration.

[0298] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intracavitary, intraarterially, intrathecally and / or intranasally, or may be directly injected into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, administration of the pharmaceutical composition may be by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the pharmaceutical composition may be administered without interruption. Such uninterrupted (or continuous) administration may be achieved by a miniature pump system worn by the patient to meter the flow rate of the therapeutic agent to the patient.

[0299] In some embodiments, the present application provides a pharmaceutical combination, which may comprise an isolated Siglec-15 binding protein of the present application, a polypeptide comprising a Siglec-15 binding protein, an immunoconjugate of the present application, a nucleic acid molecule of the present application, a vector of the present application, a cell of the present application, and / or a pharmaceutical composition of the present application, and may comprise a suitable active agent having a prophylactic and / or therapeutic effect.

[0300] In some embodiments, the pharmaceutical combination may comprise a first formulation comprising an isolated Siglec-15 binding protein of the present application and / or a polypeptide comprising a Siglec-15 binding protein and a first pharma- ceutically acceptable carrier, and a second formulation comprising a suitable therapeutically active substance and a second pharma- ceutically acceptable carrier.

[0301] In one aspect, the present application provides a kit. For example, the kit may comprise the isolated Siglec-binding protein of the present application, a polypeptide comprising a Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, a vector comprising a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, an immune complex comprising the isolated Siglec-binding protein of the present application, a cell comprising the above-mentioned substance, a composition of the present application, and / or a pharmaceutical combination of the present application. For example, the Siglec of the present application may be Siglec-15. In some embodiments, the kit is used to detect the presence and / or amount of Siglec-15 in a sample.

[0302] In one aspect, the present application provides a method of preparation.

[0303] In another aspect, the present application provides methods for preparing the isolated Siglec-15 binding protein and / or polypeptide comprising a Siglec-15 binding protein of the present application. These methods may comprise culturing a host cell of the present application under conditions capable of expressing the isolated Siglec-15 binding protein and / or polypeptide comprising a Siglec-15 binding protein of the present application. For example, these methods may be carried out by using a suitable medium, a suitable temperature, and a suitable culturing time, which are known to those skilled in the art.

[0304] Any method suitable for preparing monoclonal antibodies can be used to produce the antigen-binding proteins of the present application. For example, animals can be immunized with linked or native Siglec-15 or fragments thereof. Any suitable immunization method can be used, including adjuvants, immunostimulants, and repeated booster immunizations, and one or more routes can be used. For example, a hybridoma preparation method can be used, in which splenocytes are obtained from immunized mice and fused with SP2 / 0 myeloma cells, and hybridoma cell lines are screened by HAT.

[0305] Any suitable form of Siglec-15 can be used as an immunogen (antigen) to generate non-human antibodies specific to Siglec-15, and these antibodies are selected for biological activity. For example, the prime immunogen can be full-length mature human Siglec-15, can comprise a native homodimer, or a peptide containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

[0306] The chimeric human antibody can be selected from any class of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody can be an IgG antibody, and IgG1, IgG2, IgG3, or IgG4 subtypes can be used. Optimization of the constant domain sequence required to generate the desired biological activity can be achieved by screening the antibody using biological assays known in the art. Similarly, any type of light chain can be used in the compounds and methods of the present application. For example, kappa chains or variants thereof can be used in the compounds and methods of the present application.

[0307] In one aspect, the present application provides methods and uses.

[0308] For example, the present application provides a method for detecting Siglec in a sample, which may comprise administering an isolated Siglec-binding protein of the present application, administering a polypeptide comprising a Siglec-binding protein, administering a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, administering a vector comprising a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, administering an immune complex comprising the isolated Siglec-binding protein of the present application, administering a cell comprising the above-mentioned substance, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or administering a kit of the present application. In some cases, the method for detecting Siglec in a sample may be an in vitro method. In some cases, the method for detecting Siglec in a sample may be a non-therapeutic method. In some cases, the method for detecting Siglec in a sample may be a non-diagnostic method. For example, the Siglec of the present application may be Siglec-15.

[0309] For example, the present application provides a method of affecting the binding of a Siglec or a functionally active fragment thereof to its ligand, which may comprise administering an isolated Siglec-binding protein of the present application, administering a polypeptide comprising a Siglec-binding protein, administering a nucleic acid molecule encoding an isolated Siglec-binding protein of the present application, administering a vector comprising a nucleic acid molecule encoding an isolated Siglec-binding protein of the present application, administering an immune complex comprising an isolated Siglec-binding protein of the present application, administering a cell comprising the above-mentioned substance, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or administering a kit of the present application. In some cases, the method of affecting the binding of a Siglec or a functionally active fragment thereof to its ligand may be an in vitro method. In some cases, the method of affecting the binding of a Siglec or a functionally active fragment thereof to its ligand may be a non-therapeutic method. In some cases, the method of affecting the binding of a Siglec or a functionally active fragment thereof to its ligand may be a non-diagnostic method. For example, a Siglec of the present application can be Siglec-15. For example, a ligand of a Siglec of the present application can comprise leucine-rich repeat-containing protein 4C and sialylated Tn (sialyl-Tn). For example, affecting the binding of a Siglec, or a functionally active fragment thereof, to its ligand may be manifested as a decrease in suppression of immune system function by Siglec of greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 7%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater than about 150%, greater than about 200%, greater than about 250%, greater than about 300%, greater than about 350%, or greater than about 400% compared to a blank control.

[0310] For example, the present application provides a method of affecting immune cell proliferation, which may comprise administering an isolated Siglec-binding protein of the present application, administering a polypeptide comprising a Siglec-binding protein, administering a nucleic acid molecule encoding an isolated Siglec-binding protein of the present application, administering a vector comprising a nucleic acid molecule encoding an isolated Siglec-binding protein of the present application, administering an immune complex comprising an isolated Siglec-binding protein of the present application, administering a cell comprising the above-mentioned substance, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or administering a kit of the present application. For example, the Siglec of the present application may be Siglec-15. In some cases, the method of affecting immune cell proliferation may be an in vitro method. In some cases, the method of affecting immune cell proliferation may be a non-therapeutic method. In some cases, the method of affecting immune cell proliferation may be a non-diagnostic method. For example, affecting immune cell proliferation according to the present application may manifest as a reduction in the inhibition of immune cell proliferation by Siglec-15. For example, the proportion of a subpopulation of immune cells and / or the number of immune cells may be affected. For example, immune cells can include lymphocytes, peripheral blood lymphocytes, peripheral blood mononuclear cells and / or peripheral blood mononuclear cells. For example, the percentage of immune cell subpopulations can be increased, for example, CD8 + It is possible to increase the proportion of T cells, e.g., CD4 + The proportion of T cells can be increased. For example, CD8 + It is possible to increase the proportion of T cells, for example, CD4 + The proportion of T cells can be increased. For example, CD8 +The number of T cells can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to a blank control. + T cells may be expanded by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to a blank control.

[0311] For example, the present application provides a method of affecting cytokine secretion, which may comprise administering an isolated Siglec-binding protein of the present application, administering a polypeptide comprising a Siglec-binding protein, administering a nucleic acid molecule encoding an isolated Siglec-binding protein of the present application, administering a vector comprising a nucleic acid molecule encoding an isolated Siglec-binding protein of the present application, administering an immune complex comprising an isolated Siglec-binding protein of the present application, administering a cell comprising the above-mentioned substance, administering a pharmaceutical composition of the present application, administering a pharmaceutical combination of the present application, and / or administering a kit of the present application. For example, the Siglec of the present application may be Siglec-15. In some cases, the method of affecting cytokine secretion may be an in vitro method. In some cases, the method of affecting cytokine secretion may be a non-therapeutic method. In some cases, the method of affecting cytokine secretion may be a non-diagnostic method. For example, affecting cytokine secretion may manifest as an effect on the secretion of lipopolysaccharide-induced cytokines, such as TNF-α and / or IL-6. For example, TNF-α secretion can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 150%, more than about 200%, more than about 250%, more than about 300%, more than about 350%, more than about 400% as compared to a blank control.For example, IL-6 secretion can be increased by more than about 1%, more than about 2%, more than about 3%, more than about 4%, more than about 5%, more than about 7%, more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 150%, more than about 200%, more than about 250%, more than about 300%, more than about 350%, more than about 400% as compared to a blank control.

[0312] In another aspect, the present invention provides the use of the isolated Siglec-binding protein of the present application, a polypeptide comprising a Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, a vector comprising a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, an immunoconjugate comprising the isolated Siglec-binding protein of the present application, a cell comprising the above substances, a composition of the present application, and / or a pharmaceutical combination of the present application in the manufacture of a kit. For example, the kit can be used to detect the presence and / or amount of a Siglec in a sample. For example, the Siglec of the present application can be Siglec-15.

[0313] In one aspect, the present application provides preventive and / or therapeutic use.Siglec-15 may serve as a novel target, and antibodies targeting Siglec-15 can reverse the suppression of immune system function caused by Siglec-15.Available data may show that antibodies targeting Siglec-15 can activate immune response, and thus antibodies against Siglec-15 can be developed as drugs for preventing and / or treating disease and / or pathology, or antibodies against Siglec-15 can be used in methods for preventing and / or treating disease and / or pathology.

[0314] In another aspect, the present application provides the use of the isolated Siglec-binding protein of the present application, a polypeptide comprising a Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, a vector comprising a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, an immunoconjugate comprising the isolated Siglec-binding protein of the present application, a cell comprising the above substances, a composition of the present application, a pharmaceutical combination of the present application, and / or a kit of the present application in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition. For example, the disease and / or condition of the present application can be an immune-related disorder and / or a proliferation-related disorder. For example, the disease and / or condition of the present application can be a disease associated with aberrant expression or dysfunction of Siglec. For example, the disease and / or condition of the present application can be a Siglec-positive tumor. For example, the disease and / or condition of the present application can be a tumor with high expression of Siglec. In the present application, the disease and / or condition can be a Siglec-related disease and / or condition. For example, the Siglec of the present application can be Siglec-15.

[0315] In the present application, the disease and / or condition may include a tumor. In the present application, the disease and / or condition may include a condition involving excessive growth or proliferation of cells. In the present application, the disease and / or condition may include a proliferative disease, a neoplastic disease, and / or an immune disease. For example, the tumor of the present application may be a tumor and / or an infectious disease associated with aberrant expression or dysfunction of a Siglec protein. For example, the tumor of the present application may be a Siglec-positive tumor and / or a tumor with high expression of Siglec. In the present application, the tumor may include a solid tumor. In the present application, the disease and / or condition may include a tumor of the digestive tract. In the present application, the disease and / or condition may include an intestinal tumor. In the present application, the disease and / or condition may include colon cancer. For example, the Siglec of the present application may be Siglec-15.

[0316] In another aspect, the present application provides an isolated Siglec-binding protein of the present application, a polypeptide comprising a Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, a vector comprising a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, an immunoconjugate comprising the isolated Siglec-binding protein of the present application, a cell comprising the above substances, a composition of the present application, a pharmaceutical combination of the present application, and / or a kit of the present application for the prevention and / or treatment of a disease and / or a pathology. For example, the disease and / or pathology of the present application can be an immune-related disorder and / or a proliferation-related disorder. For example, the disease and / or pathology of the present application can be a disease associated with abnormal expression or dysfunction of Siglec. For example, the disease and / or pathology of the present application can be a Siglec-positive tumor. For example, the disease and / or pathology of the present application can be a tumor with high expression of Siglec. In the present application, the disease and / or pathology can be a Siglec-related disease and / or pathology. For example, the Siglec of the present application can be Siglec-15.

[0317] In the present application, the disease and / or condition may include a tumor. In the present application, the disease and / or condition may include a condition involving excessive growth or proliferation of cells. In the present application, the disease and / or condition may include a proliferative disease, a neoplastic disease, and / or an immune disease. For example, the tumor of the present application may be a tumor and / or an infectious disease associated with aberrant expression or dysfunction of a Siglec protein. For example, the tumor of the present application may be a Siglec-positive tumor and / or a tumor with high expression of Siglec. In the present application, the tumor may include a solid tumor. In the present application, the disease and / or condition may include a tumor of the digestive tract. In the present application, the disease and / or condition may include an intestinal tumor. In the present application, the disease and / or condition may include colon cancer. For example, the Siglec of the present application may be Siglec-15.

[0318] In another aspect, the present application provides a method for preventing and / or treating a disease and / or condition, which may comprise administering to a subject in need thereof an isolated Siglec-binding protein of the present application, a polypeptide comprising a Siglec-binding protein, a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, a vector comprising a nucleic acid molecule encoding the isolated Siglec-binding protein of the present application, an immunoconjugate comprising the isolated Siglec-binding protein of the present application, a cell comprising the above substances, a composition of the present application, a pharmaceutical combination of the present application, and / or a kit of the present application. For example, the disease and / or condition of the present application may be an immune-related disorder and / or a proliferation-related disorder. For example, the disease and / or condition of the present application may be a disease associated with aberrant expression or dysfunction of Siglec. For example, the disease and / or condition of the present application may be a Siglec-positive tumor. For example, the disease and / or condition of the present application may be a tumor with high expression of Siglec. In the present application, the disease and / or condition may be a Siglec-related disease and / or condition. For example, the Siglec of the present application can be Siglec-15.

[0319] In the present application, the disease and / or condition may include a tumor. In the present application, the disease and / or condition may include a condition involving excessive growth or proliferation of cells. In the present application, the disease and / or condition may include a proliferative disease, a neoplastic disease, and / or an immune disease. For example, the tumor of the present application may be a tumor and / or an infectious disease associated with aberrant expression or dysfunction of a Siglec protein. For example, the tumor of the present application may be a Siglec-positive tumor and / or a tumor with high expression of Siglec. In the present application, the tumor may include a solid tumor. In the present application, the disease and / or condition may include a tumor of the digestive tract. In the present application, the disease and / or condition may include an intestinal tumor. In the present application, the disease and / or condition may include colon cancer. For example, the Siglec of the present application may be Siglec-15.

[0320] In this application, administration can be performed in a variety of ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal.

[0321] Without being bound by any theory, the following examples are intended only to illustrate the binding proteins, methods of preparation, uses, etc. of the present application, and are not intended to limit the scope of the present application. EXAMPLES

[0322] Example 1 Preparation of recombinant cell lines An expression plasmid was constructed with the extracellular domain of human Siglec-15 (having the amino acid sequence described in Uniprot, Q6ZMC9, 20-263) after codon optimization and the transmembrane domain of platelet-derived growth factor receptor (PDGFR) (having the amino acid sequence described in P09619, 513-561); a lentivirus was packaged with the expression plasmid to infect 293T cells; the expression of Siglec-15 was detected by antibiotic pressure screening; the positive clones were selected and expanded to obtain a recombinant cell line human S15ECD-293T that overexpresses the amino acid sequence of the extracellular domain of human Siglec-15. Monkey S15ECD-293T can be produced by referring to the procedure similar to that described above.

[0323] Full-length mouse Siglec-15 (having the amino acid sequence set forth in Uniprot, A7E1W8) was linked to the full-length DAP12 amino acid sequence (O54885) (having the amino acid sequence set forth in SEQ ID NO: 41, GSGEGRGSLLTCGDVEENPGP) by T2A, codon-optimized, and an expression plasmid was constructed according to conventional methods; lentivirus was packaged with the expression plasmid to infect CT26 cells to generate a recombinant cell line mouse S15-CT26 overexpressing full-length mouse Siglec-15.

[0324] Example 2 Screening and maturation of Siglec-15 binding proteins Phage screening for Siglec-15 binding proteins Using the antibody library, two liquid phase phage screenings were performed against human Siglec-15 antigen. 13 Cfu of library phages were taken out, pre-bound with 50 ml of streptavidin magnetic beads for 30 min, and non-specifically bound phages were removed by specific adsorption using a magnetic rack. This procedure was repeated three times, and the mixture of the background-removed phages, 10 μg of biotinylated human Siglec-15 protein (abbreviated as HS-15 biotin or human S-15 biotin, Acrobiosystems, catalog number: SG5-H82E9), and 150 μg of streptavidin magnetic beads was incubated at 37 °C for 15 min. The mixture was washed repeatedly 14 times with PBST to remove unbound phages. Phages specifically bound to Siglec-15 were eluted with 100 mM hydrochloric acid, neutralized with Tris-HCl at pH 11, and infected into exponentially growing E. coli SS320, and then 1 h after infection, helper phage M13KO7 was added at a final concentration of 1.5 × 10 9 cfu was added to each well and the mixture was incubated overnight at 32°C. The next day, the mixture was centrifuged at 15000g for 10 min to collect the supernatant, and 1 / 5 volume of 20% PEG / NaCl solution was added to precipitate at 4°C for 1 h; the precipitate was obtained by centrifugation at 15000g for 20 min and resuspended in 1 ml of 1x PBS for the second panning, the dose of which was the same as the first, except that the dose of antigen was reduced by half each time. The enrichment rate of the second panning was determined by phage enzyme-linked immunosorbent assay (ELISA). Human Siglec-15 protein at a concentration of 2 ng / μL was added at 100 μL / well and coated overnight at 4°C. The enriched phages from the second panning were diluted to 1×10 11cfu / well and the mixture was incubated at 37° C. for 40 min. Unbound phage were eluted with PBST, 100 μL / well of HRP-conjugated mouse anti-FLAG monoclonal antibody (Sigma, 8592) at 1:5000 dilution was added, the mixture was incubated at 37° C. for 30 min, unbound phage were eluted with PBST, alkaline phosphatase color development solution was added, and absorbance was read at 450 nm in an ELISA plate reader. The results are shown in Table 1.

[0325] [Table 1]

[0326] Yeast display screening of Siglec-15 binding proteins As shown in FIG. 1, according to the construction method of yeast display of the present application using the plasmid after panning of phage antibody library as a template, primers were designed to perform polymerase chain reaction (PCR) to amplify single-chain antibody (scFv) genes, and the PCR-amplified scFv gene fragments were recovered and co-transformed with yeast display plasmid into Saccharomyces cerevisiae strain EBY100 (available from ATCC), and the scFv genes were inserted into the yeast display plasmid by homologous recombination in Saccharomyces cerevisiae, thereby displaying the single-chain antibody on the surface of the yeast cell wall. The yeast display single-chain antibody library was designated JYYDL020. Library JYYDL020 was selected and cultured overnight in 100 mL of SD-Trp medium (Clontech, Cat. No. 630308) at 30°C and 225 rpm; 1.0×10 8 The bacteria were removed and resuspended in 20 mL of YPGP liquid medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na2HPO4, 0.86% NaH2PO4·H2O), incubated at 20°C and 225 rpm for 24 h, and stored in a refrigerator at 4°C for further use.

[0327] After inducing the library, the OD of the bacterial solution 600 Measure 1OD to 1.0 x 10 7 Converted to cells, this is 4.0 x 10 8 The cells were harvested and subjected to a first round of flow cytometry sorting to enrich for the HS-15 biotin-bound positive cell population. 7 The cells were harvested and subjected to a second round of negative selection against an irrelevant antigen to remove non-specifically bound cell populations. After selection, an appropriate amount of cells was seeded onto SD-Trp solid medium and cultured at 30°C for 3 days.

[0328] Identification of Siglec-15-binding protein yeast monoclonal antibodies One 48-well plate was selected from the first screening product of JYYDL020 antibody library for sequencing and designated as Y11; two 48-well plates were selected from the second screening product of JYYDL020 for sequencing and designated as Y14-Y15. After sequence analysis, single-chain antibodies with a single sequence were obtained, and the corresponding yeast monoclones were analyzed by flow cytometry staining according to Table 2.

[0329] [Table 2]

[0330] In Scheme 1, the intensity of HS-15 biotin bound to the cell population can be reflected by the PE mean fluorescence signal intensity (MFI). Similarly, in Scheme 2, Scheme 3 and Scheme 4, the non-specific binding levels of mouse S-15 biotin (Acrobiosytems, Catalog No.: SG5-M52H7, biotinylated) and monkey S-15-Fc (Acrobiosytems, Catalog No.: SG5-C5253) can also be evaluated, and the results are shown in Table 3.

[0331] [Table 3]

[0332] According to the staining results, clones that did not bind to human, monkey, and mouse were eliminated, and the final obtained sequence of a single clone was constructed into a plasmid for expression of Siglec-15 binding protein.

[0333] Expression of Siglec-15 binding proteins The resulting full-length light and heavy chain protein sequences of the binding protein were codon-optimized, respectively, and gene synthesis was performed on the codon-optimized DNA fragment (GENEWIZ), and the synthesized gene fragment was cloned into the expression vector pcDNA3.4 (Life Technologies) in the form of IgG1. After amplification of the expression plasmid and plasmid extraction, ExpiCHO cells (ThermoFisher Scientific, A29133) were co-transfected with the two plasmids, and transient antibody expression was performed according to the supplier's ExpiCHO expression system approach, the procedure being as follows: ExpiCHO cells were cultured at 36.5 °C and 8% carbon dioxide concentration at 6 × 10 in a total culture volume of 25 ml of culture medium. 6The cells were cultured to a density of 1000 / mL, and 10 μg of antibody light chain and heavy chain expression plasmids were transfected into the cells using ExpiFecta transfection reagent. One day after transfection, 150 μL of each of 4 mL of ExpiCHO enhancer and ExpiCHO adjuvant were added to the cultured cells, and the culture was continued for 9 days. The supernatant was obtained by centrifugation at 3500 rpm and 4°C. Mix magnetic beads (AmMagTM Protein A, Genscript, L00695) with the Siglec-15 binding protein expression supernatant of the present application, incubate the mixture at room temperature for 2 hours, wash twice with PBS, discard the supernatant, add an appropriate amount of elution buffer (Protein G or A Sefinose TM Elution Buffer, Sangon, C600481), mix the mixture thoroughly and then place it on a test tube rack for 5 minutes of static incubation, during the incubation period, resuspend the magnetic beads 2-3 times, repeat the elution twice, and after elution, add an appropriate amount of neutralizing solution 1M Tris-HCl, pH 7.5 (Sangon, B548124) quickly to neutralize for subsequent use to obtain purified Siglec-15 binding protein, and the information is shown in Table 4.

[0334] [Table 4]

[0335] The purified Siglec-15 binding proteins were subjected to affinity assays, physicochemical characterization and cell binding, as well as in vitro functional activity assays, followed by affinity maturation.

[0336] Construction of affinity maturation mutant libraries The obtained Siglec-15 binding protein was subjected to affinity maturation to increase its affinity for human Siglec-15. A mutation library for each antigen-binding determinant (CDR) was constructed by randomly mutating amino acids in the CDR sites of the Siglec-15 binding protein of the present application, and high-throughput screening of sequences with strong antigen-specific binding ability was performed using yeast display technology. The amino acid sequences of the light and heavy chain variable regions of the Siglec-15 binding protein of the present application can be coded according to the Chothia numbering scheme, and the CDRs can be defined according to Chothia. For the light chain variable regions CDR1, CDR1, CDR3 and the heavy chain variable regions CDR1, CDR2, and CDR3 of the antibody molecule, NNK (N represents any nucleotide, K represents G or T) mutation primers were designed to carry out polymerase chain reaction (PCR) to amplify each CDR mutation library gene fragment, and each CDR mutation library gene fragment and yeast display plasmid were transformed into Saccharomyces cerevisiae strain EBY100, respectively, so that each CDR mutation library was displayed on the yeast surface in the form of Fab. Meanwhile, the parent sequence of the present Siglec-15 binding protein was also displayed on the yeast surface in the form of Fab, which was used as a control.

[0337] Screening of affinity maturation mutant libraries The library was cultured and induced, then 1 × 10 9 Cells were removed and a first round of enrichment was performed using a magnetic bead sorting system. Cells from each library were resuspended in 1x PBSA containing 1 nM HS-15 biotin and incubated for 30 min. After washing, cells were mixed with anti-biotin beads and incubated for 10 min. Positive cells were collected by magnetic column. Positive cells were cultured again and induced, and then 3.0 x 10 7The cells were taken out of each well and subjected to a second flow cytometry selection using 1 nM HS-15 biotin to recover cell populations with high display levels and strong binding to antigen; after the second selection, after incubation with 0.1 nM HS-15 biotin antigen, the mixture was washed with 10 mL of 1×PBSA at room temperature for 2 hours, and then subjected to a third selection; after selection, the cells were seeded on SD-Trp, Leu solid medium and cultured at 30° C. for 3 days. A single clone was picked and subjected to sequencing analysis to obtain a single sequence with mutations in each CDR of the light chain and heavy chain.

[0338] The resulting mutant plasmids for each CDR of the light and heavy chains were mixed with the parent plasmid, and the mixture was co-transformed into a yeast strain to construct a combinatorial library of light and heavy chain mutations designated JYYDL062. After culturing and inducing the library, 2.0×10 7 The cells were harvested and subjected to flow cytometry sorting using 0.1 nM HS-15 biotin to recover cell populations with high display levels and strong avidity for antigen; after sorting, the cells were plated and expanded, and single clones were picked for sequencing analysis.

[0339] Identification of a single clone After sequencing analysis, a single clone with a single sequence was identified by flow cytometry staining, and each was incubated with 1 nM HS-15 biotin for 30 minutes, and washed with 1 mL of 1×PBSA at room temperature for 2 hours; after flow cytometry analysis, the fluorescence intensity of different clones that bind to antigen was compared. Based on the staining results, clones with significantly improved binding strength to HS-15 biotin were selected for further evaluation, and the evaluation results and sequences are shown in Tables 5A, 5B, 5C, and 5D.

[0340] [Table 5]

[0341] [Table 6]

[0342] [Table 7]

[0343] [Table 8]

[0344] Expression and affinity assay of Siglec-15 binding protein affinity matured antibodies The Siglec-15 binding protein of the present application was expressed by the above-mentioned ExpiCHO expression system of the present application and purified to obtain the purified Siglec-15 binding protein, the information of which is shown in Table 6.

[0345] [Table 9]

[0346] Example 3: Affinity assay of Siglec-15 binding proteins The affinity of the present Siglec-15 binding protein to human Siglec-15 (Acro, Catalog Number: SG5-H52H3) was determined using Octet RED96e (Fortebio), and both the antigen and the present Siglec-15 binding protein were diluted in 1×PBST (1×PBS: Sangon, B548117-0500; 0.02% Tween 20: Sigma-aldrich, P1379). The antigen is used at a concentration of 30 nM, and the present Siglec-15 binding protein is used at a concentration of 33.3 nM.

[0347] Loading test samples (Octet Data Acquisotion 11.1.0.11) First, samples were added to a 96-well plate (Greiner bio-one, 655209) at 200 μL / well in the system, and the software parameters were set to set the plate temperature at 30° C. and the standard kinetic signal collection frequency was 5.0 Hz. Next, the AHC sensor (Fortebio, catalog number: 18-0015) was pre-wetted with 1x PBST for 10 minutes, and then the sample was loaded into the instrument for detection, and each cycle included the following steps: 1) soak the sample in the buffer for 60 seconds; 2) detect whether the antigen exhibits non-specific binding to the sensor; 3) regenerate in 10 mM glycine solution pH 1.7; 4) soak in the buffer for 60 seconds; 5) fix the Siglec-15 binding protein of the present application to the sensor for 20 seconds; 6) soak the sensor in the buffer for 180 seconds; 7) allow the antigen to bind to the Siglec-15 binding protein of the present application for 180 seconds; 8) allow the antigen and the Siglec-15 binding protein of the present application to dissociate for 10 minutes; 9) regenerate the sensor.

[0348] 4. Data Analysis The equilibrium dissociation constant (K D ) was determined by determining the association rate constant (kon) and dissociation rate constant (kdis) of the antigen-Siglec-15 binding protein of the present application in a 1:1 binding mode using Data Analysis 12.0 software fromx Fortebio. The results are shown in Tables 7A, 7B, and 7C below.

[0349] [Table 10]

[0350] [Table 11]

[0351] [Table 12]

[0352] Example 4 Assay of cell-binding activity of Siglec-15 binding proteins The purified Siglec-15 binding protein of the present application was characterized by flow cytometry for its function of binding to the human S15ECD-293T cells of the present application. Human S15ECD-293T cells prepared by the method of the present application were expanded to 90% confluency in 15 cm cell culture dishes, the medium was completely aspirated with a pipette, the cells were washed once with PBS buffer (Thermo Fisher, 10010049), then treated with enzyme-free cell dissociation buffer (Versene solution, Thermo Fisher, 15040066) and harvested. The cells were washed twice with PBS buffer, counted, and the desired amount of cells was removed and centrifuged, and the cells were collected at 2 × 10 6 The cells were resuspended in FACS buffer (PBS containing 2% FBS) to cells / ml. After incubation at room temperature for 15 minutes, the cell suspension was added to a U-shaped 96-well plate (Corning, 3798) at 50 μL / well. The Siglec-15 binding protein of the present application was serially diluted in FACS buffer, and the diluted solution was added to the above 96-well plate at 50 μL / well and incubated at 4°C for 1 hour. The incubated mixture was centrifuged to discard the supernatant, the cells were washed once with FACS buffer, and 100 μL of fluorescent (Alexa 488)-labeled secondary antibody (Thermo Fisher, A-11013) was added to each well and incubated at 4°C for 1 hour. The cells were washed once with FACS buffer, and 100 μL of fixative (4% (v / v) paraformaldehyde) was added to each well to resuspend the cells, and after 10 minutes the cells were washed twice with FACS buffer. The cells were resuspended in 30 μL / well of FACS buffer and detected and analyzed by flow cytometry Intellicyte Plus (Sartorius). The function of purified anti-human Siglec-15 binding protein in binding to mouse S15-CT26 cells and monkey S15ECD-293T cells was identified in a similar manner.

[0353] As shown in Figures 2, 3 and 4, the Siglec-15 binding protein of the present application can bind to Siglec-15 on the cell surface. Figure 2 shows the results of binding of the Siglec-15 binding protein of the present application to human Siglec-15 on the cell surface. Figure 3 shows the results of detection of binding of the Siglec-15 binding protein of the present application to monkey Siglec-15 on the cell surface. Figure 4 shows the results of detection of binding of the Siglec-15 binding protein of the present application to mouse Siglec-15 on the cell surface. EC of Ab9, Ab15 and isotype control of the present application for binding to human S15ECD-293T cells 50 Values ​​(nM) were 1.516, 0.4750, and NA (data not available), respectively; EC values ​​for the present Ab9, Ab15, and isotype control for binding to monkey S15ECD-293T cells in the present application. 50 Values ​​(nM) are 0.9224, 0.4746, and NA (data not available), respectively; EC values ​​for the present Ab9, Ab15, and isotype control for binding to mouse S15-CT26 cells are 50 Values ​​(nM) were 3.247, 1.827, and NA (data not available).

[0354] Example 5. Evaluation of physicochemical properties of Siglec-15 binding proteins The physicochemical properties of the Siglec-15 binding proteins of the present application can be determined with reference to the following methods.

[0355] EC-HPLC purity analysis (1) The sample was diluted to 1 mg / mL, mixed thoroughly, centrifuged at 12000 rpm for 5 minutes, and the supernatant was transferred to a sample bottle and placed on the HPLC sample tray. The chromatographic conditions were set as follows: column was TSK G3000SWxl; detection wavelength was 280 nm; column temperature was 25°C; sample chamber temperature was 5°C; flow rate was 0.5 mL / min. (2) The chromatography column was equilibrated with the mobile phase (200 mM phosphate buffer, pH 6.8), and the samples were injected and analyzed. Chromatography software was used for data analysis. The peak area percentage of each peak was calculated by the peak area normalization method.

[0356] HIC-HPLC analysis (1) The sample was diluted to 1 mg / ml, centrifuged, and the supernatant was removed and assayed. The chromatographic conditions were set as follows: column was MAbPac™ HIC-10; detection wavelength was 214 nm; column temperature was 30° C.; sample chamber temperature was 5° C.; flow rate was 0.8 mL / min. (3) Gradient elution was performed with mobile phase A (50 mM phosphate buffer / 1 M ammonium sulfate, pH 7.0) and mobile phase B (50 mM phosphate buffer, pH 7.0), and the retention time of the main peak was recorded.

[0357] Analysis of melting temperature (Tm) values The test sample was diluted to 1mg / mL with sample buffer, and then 13μL of test sample solution was added to a PCR tube, followed by 5μL of Protein Thermal shift buffer and 2μL of 10× staining solution according to the instructions of Protein Thermal Shift Starter Kit (ThermoFisher), resulting in a reaction volume of 20μL, and the mixture was mixed thoroughly and centrifuged at 12000rpm for 5 minutes to remove air bubbles. The test sample was placed into the PCR machine for sample analysis, and the Tm value of the sample was recorded.

[0358] iCIEF analysis The sample solution was added to the following thoroughly mixed system: 70 μL of 1% methylcellulose (MC), 80 μL of 5M urea, 8 μL of ampholyte Pharmalyte pH 3-10, 2 μL of pI marker 5.5, and 2 μL of pI marker 9.5. An appropriate volume of ultrapure water was added to the system to make 200 μL, and the mixture was thoroughly mixed and then centrifuged to remove the supernatant for analysis. After analysis, the resulting file was imported into ChromPerfect software to perform spectrum integration processing and calculate the isoelectric point and percentage of each peak. The Siglec-15 binding protein of the present application has a purity of 98% or more and a melting temperature Tm in the range of 86 ° C to 92 ° C, which can be said to have good temperature stability for the following development.

[0359] Example 6 Inhibitory effect of the Siglec-15 binding protein of the present invention on proliferation of T cells in human PBMC CD3 monoclonal antibody (OKT3) (Thermo Fisher, Catalog No.: 16-0037-85) was diluted to 0.03 μg / ml in PBS and coated on cell culture plates (Corning, Code 3599) (100 μL / well) overnight at 4° C. The next day, the present Siglec-15 binding protein was diluted in culture medium (RPMI1640+10%FBS) to a maximum final concentration of 12 μg / mL (prepared concentration 48 μg / mL), 4-fold serial dilution (3 gradient concentrations + 1 concentration of 0); human Siglec-15 Fc protein (Sino Biological Inc. Catalog No.: 13976-H02H) was diluted in culture medium to a final concentration of 5 μg / mL (prepared concentration 20 μg / mL). The coating solution in the coated plate was removed by pipetting, the plate was washed twice with PBS, and 50 μL of the prepared Siglec-15 binding protein of the present application was added to the plate, followed by 50 μL of the prepared human Siglec-15 Fc protein. Fresh human peripheral blood lymphocytes (AllCells, Catalog No.: PB004-C) were then labeled and centrifuged at 400 × g for 10 minutes, and the cell pellet was resuspended in PBS to a cell density of 1 × 10 7After adjusting to cells / mL, an equal volume of 2 μM CFSE (Thermo Fisher, Catalog No.: C34554) was added, mixed immediately after addition, and the mixture was placed in a 37°C water bath for 5 min and terminated with 10 volumes of PBS+10% FBS. The cells were then centrifuged at 400 × g for 10 min, washed twice with PBS+10% FBS, and resuspended in medium at 2 × 10 6 The CFSE-labeled cells were then added to the culture plate at 100 μL / well, i.e., 2 × 10 cells per well. 5 cells / well; the cell culture plate was incubated in a 37°C, 5% carbon dioxide cell incubator for 3 days, the cells were centrifuged at 300×g for 5 min, washed once with pre-chilled PBS+2% FBS, the cell pellet was resuspended in 100 μL PBS+2% FBS, 2 μL of Fc blocking reagent was added to each well, and the plate was then placed in a 4°C refrigerator for blocking for 30 min, after which the cells were mixed and the cells in each well were divided into two aliquots, one aliquot was added with 5 μL of APC mouse anti-human CD4 antibody (BD, Catalog No.: 555349), and the other aliquot was added with 5 μL of APC mouse anti-human CD8 antibody (BD, Catalog No.: 555369); after 1 h incubation in a 4°C refrigerator, the cells were washed twice with pre-chilled PBS+2% FBS, then resuspended in 30 μL of PBS+2% FBS, and analyzed on an iQue flow cytometer (Intellycite Detection was performed by loading the samples into a Flow jo V10 (Sartorius Plus). Flow results were analyzed using Flow jo V10 and data were processed using GraphPad Prism software.

[0360] FIG. 5 shows the CD4 + FIG. 6 shows the detection results of the reversal of the growth inhibitory effect of Siglec-15 protein on CD8 T cells by the Siglec-15 binding protein of the present invention. + 5 and 6 show the results of detection of the proliferation-suppressing effect of Siglec-15 protein on T cells. As shown in Figs. 5 and 6, the Siglec-15-binding protein of the present invention inhibits the proliferation of CD4 T cells. + and / or CD8 +It can reverse the growth-suppressing effect of Siglec-15 protein on T cells.

[0361] Example 7 Evaluation of the antitumor activity of the present Siglec-15 binding protein in mouse tumor models CT-26.WT (available from ATCC) mouse colon cancer cells were expanded in vitro while BMDMs (bone marrow-derived macrophages) were cultured. BMDMs were extracted from the bone marrow of the femur and tibia of BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., 7-8 weeks old, female) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 20 ng / ml macrophage colony-stimulating factor (M-CSF). After 7 days, 1 × 10 BMDMs were collected from each mouse. 5 The mice were subcutaneously inoculated with 100 μg of CT-26.WT cells in a single dose. After 12 days of inoculation, the average tumor volume of the mice was 100 mm 3 When tumor volume reached 100 mg / kg, mice were randomly divided into groups of 8 mice each according to tumor volume and body weight and treated on the same day. G1 received isotype control human IgG, G2 received our Ab9, and G3 received Ab15. Each group of mice was intraperitoneally treated with 10 mg / kg twice a week.

[0362] As shown in FIG. 7, 11 days after grouping, in group G1, the mean tumor volume was 2170±269 mm 3 in group G2, the mean tumor volume was 1677 ± 191 mm 3 and the tumor inhibition rate was 23%; in group G3, the mean tumor volume was 1540 ± 437 mm 3 The tumor inhibition rate was 29%, demonstrating that the Siglec-15 binding protein of the present invention had an inhibitory effect on tumor growth.

[0363] Example 8 Enhancement of LPS-induced cytokine secretion by the Siglec-15 binding protein of the present application BALB / c mice (commercially available from Beijing Vital River Laboratory Animal Technology Co., Ltd., 7-8 weeks old, male) were randomly divided into groups of 6 mice each according to body weight. Group G1 was administered with isotype control human IgG, group G2 was administered with the present Ab9, and group G3 was administered with Ab15. Each mouse group was intraperitoneally administered with 10 mg / kg and recorded as 0 hours; 2 hours after administration, the mice were intraperitoneally injected with lipopolysaccharide (LPS) dissolved in saline at a dose of 200 μg / kg. Two hours after LPS modeling, all animals were anesthetized by inhalation of 2-5% isoflurane, and 200 μL of whole blood was collected from the orbit; 6 hours after LPS modeling, all animals were anesthetized by inhalation of 2-5% isoflurane, and the maximum amount of peripheral blood was collected from the orbit, and then left at room temperature for 30-60 minutes, and centrifuged at 2000 g for 10 minutes at 4 °C to separate the serum for detecting cytokines TNF-α and IL-6. All experimental animals were euthanized with carbon dioxide overdose.

[0364] FIG. 8 shows the results of detecting whether the Siglec-15 binding protein of the present application affects the secretion level of TNF-α (TNF-alpha) 2 hours after LPS injection. FIG. 9 shows the results of detecting whether the Siglec-15 binding protein of the present application affects the secretion level of TNF-α (TNF-alpha) 6 hours after LPS injection. FIG. 10 shows the results of detecting whether the Siglec-15 binding protein of the present application affects the secretion level of IL-6 2 hours after LPS injection. FIG. 11 shows the results of detecting whether the Siglec-15 binding protein of the present application affects the secretion level of IL-6 6 hours after LPS injection. As shown in FIGS. 8, 9, 10, and 11, 2 hours after LPS injection, TNF-α (TNF-alpha) was significantly increased in group G2 compared to the isotype control group, and basically returned to the same level after 6 hours; 2 hours after LPS injection, IL-6 was significantly increased in all treatment groups compared to the isotype control group, and returned to the basal level after 6 hours. The present Siglec-15 binding protein was shown to promote the release of inflammatory factors in an LPS-induced acute inflammation model.

[0365] The foregoing detailed description is provided for the purposes of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently enumerated embodiments described in the present invention are obvious to those skilled in the art and are reserved within the scope of the appended claims and their equivalent solutions.

Claims

1. comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:14; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:3, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:12, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:11, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13 or SEQ ID NO:14; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:8, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:3, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:4, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:6, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; or comprising an HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:3, an HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:11, and an LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:13; An antibody or antigen-binding fragment capable of specifically binding to a sialic acid-binding immunoglobulin-like lectin (Siglec) or a functionally active fragment thereof.

2. Comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:2, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:14; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:3, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:12, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:8, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:11, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13 or SEQ ID NO:14; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:2, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:2, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:8, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:3, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:4, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13; comprising an HCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:5, an HCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as shown in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as shown in SEQ ID NO:13; comprising an HCDR3 consisting of the amino acid sequence as set forth in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence as set forth in SEQ ID NO:6, an HCDR1 consisting of the amino acid sequence as set forth in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence as set forth in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence as set forth in SEQ ID NO:10, and an LCDR1 consisting of the amino acid sequence as set forth in SEQ ID NO:13; or comprising an HCDR3 consisting of the amino acid sequence shown in SEQ ID NO:1, an HCDR2 consisting of the amino acid sequence shown in SEQ ID NO:3, an HCDR1 consisting of the amino acid sequence shown in SEQ ID NO:7, an LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence shown in SEQ ID NO:11, and an LCDR1 consisting of the amino acid sequence shown in SEQ ID NO:13; 2. The antibody or antigen-binding fragment of claim 1.

3. and / or comprising a VH comprising an amino acid sequence as set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:37; and / or A VL comprising an amino acid sequence as set forth in SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:38; 3. An antibody or antigen-binding fragment according to claim 1 or 2.

4. comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:23, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:30; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:25, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:32; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:37, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:38; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:23, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:24, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:25, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:26, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:27, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH comprising the amino acid sequence as set forth in SEQ ID NO:28, and a VL comprising the amino acid sequence as set forth in SEQ ID NO:29; or VH comprising the amino acid sequence as set forth in SEQ ID NO: 25, and VL comprising the amino acid sequence as set forth in SEQ ID NO: 31; An antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:23, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:30; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:25, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:32; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:37, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:38; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:23, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:24, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:25, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:26, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:27, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29; comprising a VH consisting of the amino acid sequence as set forth in SEQ ID NO:28, and a VL consisting of the amino acid sequence as set forth in SEQ ID NO:29; or Comprising a VH consisting of the amino acid sequence as shown in SEQ ID NO: 25, and a VL consisting of the amino acid sequence as shown in SEQ ID NO: 31; An antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. and / or comprising an antibody heavy chain constant region; preferably a heavy chain constant region derived from a human antibody; more preferably a heavy chain constant region derived from an IgG; more preferably a heavy chain constant region derived from an IgG1; and / or An antibody light chain constant region; preferably a light chain constant region derived from a human antibody; more preferably a constant region derived from Igκ; An antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody is a monoclonal antibody.

8. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the antibody is selected from one or more of the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

9. The antigen-binding fragment may be a Fab, Fab', Fv fragment, F(ab') ２ , F(ab) ２ 9. The antibody or antigen-binding fragment of any one of claims 1 to 8, selected from one or more of the group consisting of: scFv, di-scFv, VHH, and dAb.

10. The Siglec comprises Siglec-15, preferably the Siglec comprises human Siglec, monkey Siglec, and / or mouse Siglec, preferably the Siglec-15 or a functional fragment thereof comprises Siglec-15 or a functional fragment thereof expressed on a cell; optionally the antibody or antigen-binding fragment has the ability to bind to Siglec-15 or a functional fragment thereof with a K D value of about 1.56E-10 M or less; the antibody or antigen-binding fragment has the ability to bind to Siglec-15 or a functional fragment thereof with an EC 50 value of about 3.25 nM or less; the antibody or antigen-binding fragment has the ability to affect immune cell proliferation, preferably affecting immune cell proliferation comprises reducing Siglec-15 associated immune cell proliferation inhibition, more preferably said immune cells comprise human derived immune cells, more preferably said immune cells comprise lymphocytes, peripheral blood lymphocytes, T cells, CD4 + cells and / or CD8 + cells; the antibody or antigen-binding fragment has tumor-inhibiting ability, preferably said tumor-inhibiting ability comprises affecting tumor volume; the antibody or antigen-binding fragment has the ability to inhibit tumors with a tumor volume inhibition rate of about 23% or greater; and / or the antibody or antigen-binding fragment has the ability to affect cytokine secretion, preferably said secretion comprising in vivo, ex vivo and / or in vitro secretion, preferably said cytokine secretion comprising lipopolysaccharide-induced cytokine secretion, preferably said cytokine comprising the inflammatory factors, TNF-α and / or IL-6; An antibody or antigen-binding fragment according to any one of claims 1 to 9.

11. A nucleic acid molecule encoding an antibody or antigen-binding fragment described in any one of claims 1 to 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. An immune complex comprising an antibody or antigen-binding fragment described in any one of claims 1 to 10.

14. A cell comprising and / or expressing an antibody or antigen-binding fragment of any one of claims 1 to 10, a cell comprising the nucleic acid molecule of claim 11, a cell comprising the vector of claim 12, and / or a cell comprising the immune complex of claim 13.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the vector of claim 12, the immune complex of claim 13, and / or the cell of claim 14, and optionally a pharma- ceutical acceptable vehicle.

16. A pharmaceutical combination comprising the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the vector of claim 12, the immune complex of claim 13, the cell of claim 14, and / or the pharmaceutical composition of claim 15.

17. A kit comprising an antibody or antigen-binding fragment according to any one of claims 1 to 10, a nucleic acid molecule according to claim 11, a vector according to claim 12, an immune complex according to claim 13, a cell according to claim 14, a pharmaceutical composition according to claim 15, and / or a pharmaceutical combination according to claim 16, preferably for detecting the presence and / or amount of Siglec in a sample, more preferably wherein the Siglec comprises Siglec-15.

18. A method for preparing an antibody or antigen-binding fragment described in any one of claims 1 to 10, comprising culturing a cell described in claim 14 under conditions capable of expressing the antibody or antigen-binding fragment.

19. 17. A method for detecting Siglec in a sample, comprising administering an antibody or antigen-binding fragment according to any one of claims 1 to 10, administering a nucleic acid molecule according to claim 11, administering a vector according to claim 12, administering an immunoconjugate according to claim 13, administering a cell according to claim 14, administering a pharmaceutical composition according to claim 15, administering a pharmaceutical combination according to claim 16 and / or using a kit according to claim 17, wherein preferably said Siglec comprises Siglec-15.

20. The antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the vector of claim 12, the immunoconjugate of claim 13, the cell of claim 14, the pharmaceutical composition of claim 15, the pharmaceutical combination of claim 16, and / or the kit of claim 17 for the prevention and / or treatment of a disease and / or condition, preferably wherein the disease and / or condition comprises a tumor, preferably wherein the tumor comprises a Siglec-associated tumor, preferably wherein the Siglec comprises Siglec-15, optionally wherein the disease and / or condition comprises a solid tumor, preferably colon cancer.

21. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the immunoconjugate according to claim 13, the cell according to claim 14, the pharmaceutical composition according to claim 15, the pharmaceutical combination according to claim 16 and / or the kit according to claim 17 in the manufacture of a medicament for the prevention and / or treatment of a disease and / or pathology, comprising Preferably, said disease and / or condition comprises a tumor; Preferably, the tumor comprises a Siglec-associated tumor; Preferably, the Siglec comprises Siglec-15; Optionally, the disease and / or condition comprises a solid tumor, preferably colon cancer.

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